Biologically active compounds and methods of constructing and using the same

ABSTRACT

A method of constructing biologically active compounds which mimic the biological activity of the biologically active protein block the activity of the biologically active protein is disclosed. A method of identifying specific and discrete portions of pathogen antigens which either serve as epitopes for neutralizing antibodies or which are involved in pathogen binding to host cell receptors is disclosed. A method of constructing biologically active compounds which compete with cellular receptors for binding to either biologically active proteins or pathogen antigens is disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a continuation of U.S. Ser. No. 07/940,654 filedSep. 3, 1992, allowed as U.S. Pat. No. 5,637,677, which is aContinuation-In-Part Application of U.S. patent application Ser. No.07/702,833, filed May 20, 1991, abandoned, which is a ContinuationApplication of U.S. patent application Ser. No. 07/326,328, filed Mar.21, 1989, abandoned, which was a Continuation-In-Part Application ofU.S. patent application Ser. No. 07/074,264, filed Jul. 16, 1987,abandoned. This Application is a Continuation-In-Part Application ofU.S. patent application Ser. No. 07/462,542, filed Jan. 9, 1990,abandoned, which is a Divisional Application of U.S. patent applicationSer. No. 07/074,264, filed Jul. 16, 1987, abandoned. This Application isa Continuation-In-Part Application of U.S. patent application Ser. No.07/648,303, filed Jan. 25, 1991, abondoned, which is a File WrapperContinuation Application of U.S. patent application Ser. No. 07/074,264,filed Jul. 16, 1987, abandoned. This Application is aContinuation-In-Part Application of U.S. patent application Ser. No.07/685,881, filed Apr. 15, 1991, abandoned, which is a ContinuationApplication of U.S. patent application Ser. No. 07/574,391, filed Aug.27, 1990, abandoned which was a File Wrapper Continuation Application ofU.S. patent application Ser. No. 07/194,026 filed May 13, 1988, allowedas U.S. Pat. No. 4,962,510 which was a Continuation-In-Part Applicationof U.S. patent application Ser. No. 07/074,264, filed Jul. 16, 1987,abandoned. This Application is a Continuation-In-Part Application ofU.S. patent application Ser. No. 07/583,626, filed Sep. 14, 1990,abandoned. Each of the above listed patent applications is incorporatedherein by reference.

ACKNOWLEDGMENT OF GOVERNMENT RIGHTS

This invention was made with Government support under grant 5R01EY08191awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods of identifying portions ofproteins involved in protein-protein interactions, to methods ofconstructing biologically active peptides involved in protein-proteininteractions, and to biologically active peptides.

BACKGROUND OF THE INVENTION

Protein binding or protein-protein interactions can be broadly definedas the discrete interaction of the surface of one protein with thesurface of another protein. Such discrete interaction arises whenresidues of one protein are proximally located to residues of anotherprotein and attractive forces between the residues such as vander Waalsforces, ionic bonds and hydrogen bonds exist. Specific protein-proteininteractions which occur in higher living organisms include but are notlimited to those in which involve: a receptor-binding protein binding toa receptor; a pathogen antigen binding to a host cell receptor; proteininteractions at cellular attachment sites; and, adhesion proteinsinteractions.

Examples of receptor-binding proteins, hereinafter also referred to asligands, include cytokines, hormones and growth factors. These proteinsbind to receptors on cells and cause changes in cellular activity orfunction. For example, cytokines are a variety of proteins which arecellular messengers, each cytokine having a specific effect upon a cell.Likewise, hormones and growth factors are also messengers which affectthe function and activity of cells.

Pathogens are infectious organisms, such as bacteria, fungi, parasites,and viruses and, additionally, neoplasms, all of which express specificantigens. Such typically, there are specific sites on antigens,hereinafter referred to as binding epitopes or epitopes, which bind to acomplementary portion of a cellular protein called a receptor site.

A great deal of effort has been expended in search of compounds whichspecifically either simulate, that is mimic, or block protein-proteininteractions in cells.

With respect to cytokines, hormones and growth factors, a great deal ofeffort has been made to purify the natural proteins from natural sourcesor to synthetically produce them by chemical means or using recombinantDNA technology. While some success has been achieved, these moleculesare quite large, difficult to handle and expensive to obtain. A greatdeal of effort is also directed at discovering synthetic ligands whicheither mimic the activity of natural proteins or which block theactivity of natural proteins. Blocking natural protein activity can beachieved by either competing for the receptor with an inactive ligand(antagonist) or by having an agent bind to the natural protein andthereby prevent it from binding to the receptor.

There is a need for synthetic peptides and/proteins which mimic theactivity of the natural biologically active proteins which interact withreceptors. Such mimicking molecules would be useful as agents to affectthe cells in the same way as the natural protein. Likewise, thediscovery of antagonists, that is, molecules which block the receptorwithout having an effect on cellular function or activity would beuseful. Furthermore, the discovery of agents which specifically interactwith biologically active proteins and thereby render them unable to bindto receptors is also desirable. Molecules that prevent binding by anatural biologically active protein to its receptor in cases where thenatural protein is believed to an agent associated with a diseasecondition or disorder are useful as drugs for preventing or treatingsuch disease conditions or disorders. A great deal of knowledge has beendeveloped in the field of immunology, including at the molecular level.

Advances in molecular biology have indicated that immunoglobulins, majorhistocompatibility complex antigens and T-cell receptors are all membersof a family of molecules referred to as the immunoglobulin superfamily.During evolution, it is likely that a single, useful gene duplicated,and its copies diverged to create related molecules with distinctfunctions. Accordingly, immunoglobulins, which are agents of humoralimmunity; T-cell receptors, which are associated with humoral as well ascellular immunity; and major histocompatibility complex molecules,involved in antigen presentation and the discrimination between self andnonself, all share homologies inherited from their common ancestor andexhibit related biological functions.

Of the members of the superfamily, the structure and function ofimmunoglobulins is best understood.

Immunoglobulin molecules consist of a constant region and a variableregion. The constant region is associated with cellular effectorfunctions whereas the variable region participates in antigenrecognition and binding.

Immunoglobulins of the most common class, IgG, consist of two heavychains and two light chains linked together by noncovalent associationsand also by covalent disulfide bonds. Each of the chains possesses aconstant as well as a variable region. In the immunoglobulin molecule,the variable region is subdivided into framework regions, which aresimilar in structure among immunoglobulins, and hypervariable,complementarity determining regions (CDRs) which participate directly inantigen binding in the immunoglobulin active site.

X-ray crystallographic studies of purified immunoglobulin molecules haveindicated that the active site is a crevice formed by the heavy andlight chain variable regions, and that the dimensions of the activesites vary among immunoglobulin molecules consequent to amino acidsequence variations (Hood et al., 1978, in “Immunology,” TheBenjamin/Cummings publishing Co., Inc., Menlo Park, p. 208). Amino acidsequence, crystallographic structure, and specially designed haptenprobes have been used in conjunction with computer analysis to elucidatethe relationship between an immunoglobulin and the antigen which itrecognizes.

Pathogens generally express antigens which are recognized by host immunesystems as foreign and become the target of an immunological response toeliminate the infectious pathogen. Pathogen antigens often bind tocellular receptors on a host's cells as part of the process of infectionof the host by the pathogen. In order to immunize the host and reducethe effectiveness of the pathogen to mount a challenge to the host, anumber of vaccination strategies have been devised.

Several strategies have been employed to develop safe, effectivevaccines against viral and bacterial pathogens. At present most vaccinesin use consist of live attenuated pathogens, killed pathogens,components of a pathogen, or modified toxins (toxoids). See Institute ofMedicine, “Vaccine Supply and Innovation”, Washington, D.C.: NationalAcademy Press (1985). While these preparations have been successfullyused for many infectious diseases, many pathogens exist where theseapproaches have not worked or have not been applicable. Certainpathogens are potentially too dangerous to contemplate the use ofattenuated or even inactivated preparations. The risk of developingcancer from immunization with certain retroviruses, or of developingacquired immunodeficiency syndrome (AIDS) from immunization with humanimmunodeficiency virus (HIV) underscores the drawbacks associated withthe use of whole virus preparations for vaccination. In addition manypathogens display a marked antigenic heterogeneity that makes effectivevaccination difficult. These considerations have led us to seekalternative method for effective immunization.

The idiotype network theory of N. K. Jerne, Ann. Immunol. (Paris)125:337-389, (1974), implies that an anti-idiotypic antibody raisedagainst a neutralizing antibody specific for a pathogen would mimic thatpathogen immunologically. Immunization with the anti-idiotype shouldresult in the development of a significant anti-pathogen response withthe elicitation of neutralizing antibodies and cell-mediated immunity.In recent years there have been several examples where this strategy hasbeen effective, including reovirus type 3. See Sharpe, A. H., et al., J.Exp. Med. 160:195-205 (1984); Kauffman, R. S., et al., J. Immunol.,131:2539-2541, (1983); and Gaulton, G. N., et al., J. Immunol.137:2930-2936. With respect to Sendai virus, see Ertl, H. C. andFinberg, R. W., Proc. Natl. Acad. Sci. USA 81:2850-2854 (1984). Forreport relating to rabies see Reagen, K. J. et al., J. Virol. 48:660-666(1983). This approach has been discussed in connection with polio virusin Uydeltaag, F.G.C.M. and Osterhaus, A.D.M.E., J. Immunol.134:1225-1229 (1985).

One of the key aspects of this approach is that a portion of theanti-idiotype mimics a portion of the pathogen antigen and induces aneutralizing response. Thus a potent anti-idiotype vaccine would seem tobe an ideal immunogen in cases where intact pathogen could not be usedor where irrelevant non-neutralizing epitopes dominate the immuneresponse. However, the practical application of anti-idiotypes asvaccine has been limited by the difficulties in making human monoclonalantibodies and in the danger of producing serum sickness by usingxenogeneic antibodies.

Another method currently under intensive investigation is the use ofsynthetic peptides corresponding to segments of the proteins frompathogenic microorganisms against which an immune response is directed.This approach has been successful in several instances including felineleukemia virus (Elder, J. H. et al., J. Virol. 61:8-15, 1987), hepatitisB (Gerin, J. L., et al., Proc. Natl. Acad. Sci. USA, 80:2365-2369 1983),Plasmodium falciparum (Cheung, A., et al., Proc. Natl. Acad. Sci. USA83:8328-8332, 1986), cholera toxin (Jacob, C. O., et al., Eur. J.Immunol. 16:1057-1062, 1986) and others. When these peptides are capableof eliciting a neutralizing immune response they appear to be idealimmunogens. They elicit a specific response and typically do not lead todeleterious effects on the host. However, it can be difficult to predictwhich peptide fragments will be immunogenic and lead to the developmentof a neutralizing response.

It would be desirable to develop immunogens that elicit a response tospecific neutralizing epitopes without causing responses to extraneousepitopes that could “dilute” the specific response or lead to harmfulimmune complex formation.

The present invention relates to a method of identifying specific linearand constrained discrete portions of a biologically active proteinsinvolved in protein-protein interactions. By identifying such specificand discrete portions, biologically active peptides can be constructedwhich mimic the biological activity of the biologically active proteinor which block the activity of the biologically active protein. Thus,biologically active peptides can be constructed which act as ligandsthat act on mammalian cells by binding to the receptor sites of thosecells to alter or affect their function or behavior, or to prevent thebinding of the natural biologically active protein to the cellularreceptor, thereby preventing the biologically active protein fromaffecting the cell.

The present invention relates to a method of identifying specific linearand constrained discrete portions of pathogen antigens which eitherserve as epitopes for neutralizing antibodies or which are involved inpathogen binding to host cell receptors. By identifying discreteportions of pathogen antigens which are neutralizing epitopes,biologically active peptides can be constructed which are useful ascomponents of vaccines against the pathogen. An effective neutralizingimmune response will be elicited in a vaccinated individual. Byidentifying discrete portions of pathogen antigens which are involved inpathogen binding to host cell receptors, biologically active peptidescan be constructed which are useful as agents which block pathogenattachment to cellular receptors. Additionally, by identifying discreteportions of pathogen antigens which are involved in pathogen binding tohost cell receptors, biologically active peptides can be constructedwhich mimic pathogen antigens and act on mammalian cells by binding tothe receptor sites of those cells to alter or affect their function orbehavior, or which prevent or alter the effect which pathogen antigenswould otherwise have upon those cells.

The present invention relates to the field of biologically activepeptides which have some shared and/or similar amino acid sequences tothe amino acid sequences of cellular receptor sites and thereby competewith such cellular receptors for binding to either biologically activeproteins or pathogen antigens. In addition, the invention relates to thefield of biologically active peptides which have some shared and/orsimilar amino acid sequences to the amino acid sequences of the ligandsurface that attaches to a cellular receptor site. The ligand mimeticpeptide can be used as a stimulant or inhibitor of that receptor. Wherethe biologically active peptide competes in pathogen/receptor binding,the biologically active peptides are useful to prevent pathogenattachment and thereby prevent infection. Where the biologically activepeptide competes in biologically active protein/receptor binding, thebiologically active peptides are useful to prevent ligand/receptorbinding and thereby prevent the effect on cellular function or behaviornormally associated with the biologically active protein/receptorbinding.

SUMMARY OF THE INVENTION

One embodiment of the invention relates to a method of constructing apeptide capable of eliciting a neutralizing immune response against apathogen in a mammal. A method of the invention comprises the steps ofidentifying a neutralizing epitope of a pathogen antigen by firstgenerating a neutralizing antibody specific for a pathogen, thengenerating an anti-idiotypic antibody specific for the neutralizingantibody and then identifying the CDR amino acid sequence of theanti-idiotypic antibody that corresponds to an amino acid sequence of apathogen antigen. Using that information, a peptide is synthesized thatcorresponds to or is identical to the portions of the antibody andantigen that correspond to each other.

Another embodiment of the invention relates to a method of immunizing ahost mammal against infection by a pathogen that comprises such anantigen by inoculating a mammal with a peptide that corresponds to theneutralizing epitope of a pathogen antigen.

Another embodiment of the invention is a method of constructing apeptide capable of preventing a pathogen or a biologically activeprotein from binding to a cellular receptor. A method of the inventioncomprises identifying an amino acid sequence of a portion of a pathogenantigen or a biologically active protein which binds to the cellularreceptor by first generating an anti-receptor antibody capable ofpreventing a pathogen or a biologically active protein from binding tothe cellular receptor and then identifying an amino acid sequence of theanti-receptor antibody that corresponds to an amino acid sequence of thepathogen antigen or the biologically active protein. Using thatinformation, a peptide is synthesized that corresponds to or isidentical to the portions of the antibody and antigen that correspond toeach other.

Another embodiment of the invention relates to a method of treating ahost mammal to prevent or reduce the severity of an infection by apathogen by constructing a peptide capable of preventing a pathogen frombinding to cellular receptor and administering the synthetic peptide toa mammal in an amount effective to prevent or reduce the likelihood thatthe pathogen will infect cells of the host.

Another embodiment of the invention relates to a method of constructinga peptide capable of preventing a pathogen or a biologically activeprotein from binding to a cellular receptor. A method of the inventioncomprises identifying an amino acid sequence of a cellular receptorwhich directly interacts with an amino acid sequence of a pathogenantigen or a biologically active protein during receptor binding byfirst generating an antibody specific for the pathogen or thebiologically active protein which is capable of preventing the pathogenor the biologically active protein form binding to the cellular receptorand then identifying an amino acid sequence of the CDR of the antibodywhich corresponds to an amino acid sequence of the cellular receptor.Using that information, a peptide is synthesized that corresponds to oris identical to the portions of the antibody and antigen that correspondto each other.

Another aspect of the invention relates to a method of constructing abiologically active peptide comprising the steps of identifying an aminoacid sequence of a biologically active portion of a biologically activeprotein which directly interacts with a cellular receptor when thebiologically active protein binds to the cellular receptor, wherein suchbinding causing an effect on an activity or function of cell. The aminoacid sequence of the biologically active portion of the biologicallyactive protein identified by first generating an anti-receptor antibodyagainst a cellular receptor, the anti-receptor antibody being capable ofeffecting an activity or function of a cell and then identifying anamino acid sequence of the CDR of the anti-receptor antibody thatcorresponds to an amino acid sequence of the biologically activeprotein. Using that information, a peptide is synthesized that iscorresponds to or is identical to the portions of the antibody andantigen that correspond to each other.

Another embodiment of the invention relates to a method of effecting oraltering activity or function of a mammalian by contacting a cell withan amount of such a biologically active peptide sufficient to effect oralter activity or function of the cell.

The invention relates to synthetic biologically active peptidescomprising or consisting essentially of amino acid sequence thatcorrespond to an amino acid sequence of an antigen or biologicallyactive protein and an amino acid sequence of an anti-idiotypic antibodyor an anti-receptor antibody.

BRIEF SUMMARY OF THE FIGURES

FIG. 1 illustrates the specific binding of 9BG5 to peptides, determinedby radioimmunoassay as noted in the experimental procedures describedhereinafter; CPM of 9BG5 bound to blank wells was subtracted from CPM of9BG5 bound to peptide coated wells; non-specific binding to peptides wascorrected for by subtracting from the value a similar value determinedfor an isotype-matched control monoclonal antibody UPC10; specific CPMof 9BG5 bound to peptide coated wells is shown using the amount of 9BG5added to each well in a final volume of 50 μl.; mean ±SD for duplicatewells is shown.

FIG. 2A and 2B illustrate the binding of V_(L)-BSA to type 3 reovirusreceptor as determined by its ability to compete for binding withanti-reovirus type 3 receptor antibody 87.92.6.; R1.1 cells (10⁷/ml)were incubated in 1% BSA in the presence or absence of 200 μg/mlV_(L)-BSA or V_(H)-BSA as indicated for 45 minutes; monoclonalantibodies were added at the concentrations noted for an additional 30minutes; the cells were washed twice and a 1:200 dilution of FITC-goatanti-mouse Fab was added for 30 minutes; the cells were washed twice andanalyzed for fluorescence intensity on a FACS analyzer; percent maximalcell staining was determined as the ratio of the percent of the cellspositive on FACS analysis at the antibody concentration noted to themaximal percent of cells judged positive at saturating doses ofmonoclonal antibody in the absence of competitors ([% positive atconcentration divided by maximal % positive] ×100); the maximal percentpositive values were as follows: 2a - 15.3%, 2b - 97%, 2c - 24%.

FIGS. 3A and 3B shows reovirus type 3 and 87.92.6 antibody inhibition ofL cell proliferation.

FIG. 4 shows inhibition of L cell proliferation by peptides.

FIGS. 5A-D show modulation of reovirus type 3 receptor by peptides.

FIG. 6 shows modulation of the reovirus type 3 receptor by peptides andantibody.

FIGS. 7A and 7B show inhibition of lymphocyte proliferation.

FIGS. 8A and 8B show peptide inhibition of con A induced lymphocyteproliferation.

FIG. 9 shows competition of binding of 9BG5 antibody to 87.92.6 antibodycoated wells in the presence of peptide inhibitors.

FIGS. 10A-C show V_(L) and variant peptide inhibition of binding ofreovirus type 3 particles to 9BG5.

FIGS. 11A and 11B show in (a) and (b) V_(L) peptide inhibition ofbinding of reovirus type 3 and variant K to L cells; FIGS. 11C and 11Dshow V_(L) variant peptide inhibition of binding of reovirus type 3 tomurine L cells.

FIGS. 12A-C illustrate specific binding of immune serum to virus-coatedplates; determined by radioimmunoassay as noted in the hereinafterdescribed experimental procedures; CPM of immune serum binding to blankwells was subtracted from CPM binding to virus coated wells; to accountfor non-specific binding to virus coated wells, a similar valuedetermined for normal mouse serum was subtracted form the valuedetermined for immune serum; specific CPM bound is shown versus thedilution of mouse serum added in a final volume of 50 μl.; the mean ±SEMof duplicate wells from groups of 3 or 4 mice is shown at each dilution.

FIG. 13A and 13B illustrate immune serum assays for viral neutralizationas described in the following section; serum was collected prior toimmunization with peptides (pre-immune or day 0), on day 20 followingthe first immunization, and on day 60; the neutralization titer wasdetermined at each time point from groups of 4 mice; the geometric meandivided by SEM of the reciprocal of the neutralization titer is shown ateach time point.

FIG. 14A and 14B illustrate plaque inhibition, determined as indicatedin the following description; plaque numbers were determined for 4 micein each group and the mean values determined; the highest dilution ofserum that produced 50% or greater plaque inhibition was determined andis shown for each time point at which serum was obtained; plaqueinhibition of both type 1 and type 3 virus is shown.

FIG. 15A and 15B shows the delayed type hypersensitivity (DTH) responseof mice to intact reovirus type 3 after immunization with peptides.

FIG. 16 shows a representational diagram of two alternate routes for thedevelopment of biologically active peptides according to the methods ofthe invention.

FIG. 17 illustrates data for mice immunized with the reovirus typesnoted by injection of 10⁷ PFU subcutaneously, or with the peptides notedat a dose of 100 μg split into two injections subcutaneously; one weeklater, mice were challenged with virus or peptides in the footpads;footpad swelling was determined as indicated in the followingdescription 48 hours after challenge; the mean +SEM for groups of miceis shown.

FIG. 18. Structural similarities in gp120 binding domain with Igsuperfamily. Complementarity determining regions (CDR) and frameworkregions (FR) of the first, second, third and fourth domains of therespective heavy (H) or light (L) chains of several antibodies exhibiteda degree of sequence homology with gp120 residues 383-455. The asterisks(*) mark residue positions of shared sequence homology between other HIVisolates and other antibodies. Crystallographic analysis of antibodiesindicates that structural characteristics of CDR regions are preservedin spite of differences in sequence among antibodies. The dash (-) belowa residue position denotes a lack of any sequence homology between anHIV isolated and an antibody. The dash (-) within a sequence denotes adeletion or insertion.

FIG. 19. Backbone representation of a proposed model for the putativebinding side of gp120. The model extends from residue 413 throughresidue 456.

FIG. 20. Comparison of cyclic and linear peptide interactions with theReo3R by inhibition of ¹²⁵-reovirus type 3 binding.

FIG. 21. Comparison of binding of antisera resulting from immunizationof rabbits with B138, 466, 1005-45, or 1029-04 peptides to gp120.

FIG. 22. Sequence homology of CD4 and L3T4 with Ig light chains of knownthree-dimensional structure. Boxed areas highlight similar sequences.Dashes (-) indicate insertions/deletions. Sequence alignment forcomparative model building of CD4 utilizes a crystallographic templatesubstituting the sequence of CD4 onto the homologous template. Thechoice of template is decided based upon the degree of sequence homologybetween a template and CD4 and the length of analogous turn/loopstructures.

FIG. 23. Rate of loss of sulfhydryls for various peptides.

FIG. 24. Binding of 9B.G5 to peptides on solid phase RIA.

FIG. 25. Inhibition of 9B.G5 - 87.92.6 interaction by cyclic peptides.

FIG. 26. Inhibition of 9B.G5 - 87.92.6 interaction by cyclic peptides.Comparison with linear peptides derived from the 87.92.6 variableregions.

FIG. 27. Inhibition of 9B.G5 - reovirus type 3 interaction by cyclicpeptides.

FIG. 28. Inhibition of 9B.G5 - reovirus type 3 interaction by cyclicpeptides. Comparison with linear and dimeric peptides derived from the87.92.6 variable regions.

FIG. 29. Inhibition of 87.92.6-Reo3R interaction by peptides.

FIG. 30. Specificity of V_(L)C₉C₁₆ peptide binding to the Reo3R.

FIG. 31. Inhibition of reovirus type 3 - Reo3R interaction by peptides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the invention, specific and discrete portions of proteinsinvolved in protein-protein interactions can be identified andbiologically active peptides can be constructed based upon the aminoacid sequences identified. The amino acid sequences of specific portionsof anti-idiotypic antibodies correspond to the amino acid sequence ofthe specific portion of the epitope of an antigen that binds to anidiotypic antibody. Likewise, the amino acid sequences of specificportions of anti-receptor antibodies correspond to the amino acidsequences of the specific portion of a ligand which interacts with thereceptor. Thus, the amino acid sequence of either the critical portionof an epitope or the biologically active portion of a biologicallyactive protein can be identified.

The attachment of proteins to one another often involves secondarystructural features such as loops or helices. The disposition ofspecific kinds of residues (aromatic and hydrophilic) allows attachmentto occur through interactions between the residues of the differentproteins. These interactions include vander Waals interactions andhydrogen bonds. The individual loops that occur in portions ofantibodies, for example, form hydrogen bonds with antigen fragments.Likewise, individual loops that occur in portions of receptor moleculesform hydrogen bonds with receptor binding proteins.

An idiotype is the set of idiotopes which are antigenic determinants.The idiotopes occur in the CDR portion of the variable region of aparticular antibody. Antigens represented by an idiotype have specificinteractions with the antibody which results in bind. Such idiotypes arecalled internal images of antigens. An anti-idiotypic antibody is anantibody is specific for the portion of another antibody that representsthe idiotope regions. The idiotype or internal image of ananti-idiotypic antibody is similar to the antigen that the idiotypicantibody recognizes.

Thus, peptides modelled from the surface of a highly variable CDR loopare used to mimic a region (loop or loop portion of an alpha helix) ofsome other protein. In some cases, more than one surface can be linked,forming dimers. In other cases, the loops are constrained withspecifically placed cysteine residues or by placement of other residueswhich permit loop closure such as through, for example, ionic bonds.

As used herein, the term “biologically active protein” refers toproteins which bind to cellular receptors and thereby alter or affectthe function or behavior of the cells, or prevent or alter the effectwhich another biologically active protein would otherwise have uponthose cells. A pathogen antigen can be a biologically active protein if,upon binding to a host cell, it alters or affects the function oractivity of a cell or prevents another agent from doing so. Otherexamples of biologically active proteins include, but are not limitedto, cytokines, hormones and growth factors.

As used herein, the term “neutralizing epitope” refers to the portion ofa pathogen antigen against which antibodies have a neutralizingactivity. That is, antibodies specific for a neutralizing epitope willrender the pathogen non-infective and/or inactive.

As used herein, the term “neutralizing antibodies” refers to antibodieswhich recognize a pathogen and render it non-infective and/or inactive.

As used herein, the term “anti-pathogen antibodies” refers to antibodieswhich recognize and bind to a pathogen, specifically a pathogen antigen.

As used herein, the term “anti-receptor antibodies” refers to antibodieswhich recognize and bind to a receptor, specifically at a receptor site.Anti-receptor antibodies are specific forms of anti-idiotypicantibodies. Anti-receptor antibodies are anti-idiotypic antibodies whichare specific for the idiotype of an immunoglobulin molecule. That is,they are specific for the portion of the immunoglobulin receptor whichinteracts with a biologically active protein.

As used herein, the term “receptor site” refers to the portion of thereceptor that interacts with a protein that binds to the receptor.

As used herein, the term “biologically active peptides” refers toproteinaceous molecules which mimic biologically active proteins orprevent the interaction between biologically active proteins andreceptors.

Biologically active peptides can be constructed which function as theepitope or mimic a biologically active protein. Alternatively,biologically active peptides can be constructed which interact withreceptors and thereby block the binding of a pathogen antigen orbiologically active protein to a receptor.

As used herein, the term “biologically active compound” refers to acompound which mimics a biologically active protein or which canotherwise interact with a receptor and thereby block the binding of apathogen antigen or biologically active protein to a receptor.Additionally, a biologically active compound can mimic an epitope of anantigen of a pathogen and elicit a neutralizing immune response in amammal. A biologically active compound may be a peptide or anon-peptidyl compound including, but not limited to, compounds whichcomprise amino acid sequences linked by non-peptide bonds. The term“compounds” as used herein refers to peptides and non-peptidylcompounds.

One having ordinary skill in the art can appreciate that biologicallyactive compounds can be synthesized which comprise amino acid sequencesfound in peptides but which are linked by non-peptide bonds. One havingordinary skill in the art can readily appreciate that the essential stepof identifying the biologically significant portion of an antigen orligand allows for the construction of compounds, peptide andnon-peptide, which mimic the function or activity of the antigen orligand.

Accordingly, the methods of the invention also relate to constructingand using biologically active compounds that are modelled based uponcorresponding amino acid sequences of antigen or ligands andanti-idiotypic or anti-receptor antibodies. The identification ofcorresponding sequences in portions of anti-idiotypic antibodies oranti-receptor antibodies and pathogen antigens or biologically activeproteins can be used in the construction of biologically activecompounds which comprise such shared amino acid sequences but which arelinked by non-peptide bonds. Furthermore, using well known techniques,such non-peptide biologically active compounds can be synthesized fromreadily available starting materials be those having ordinary skill inthe art.

As used herein, the terms “correspond” and “corresponding” refer to thelevel of shared identity between two amino acid sequences. That is, theamount of identical and conservatively substituted amino acid sequencesshared between two molecules. As used herein, two sequences correspondif, when compared, they share approximately at least 80% identical andconservatively substituted sequences of which at least about 28% areidentical sequences and between about 30-42% conservative substitutions.Generally, corresponding amino acid sequences share at least six similaramino acid residues. Corresponding sequences are often longer,comprising about 10 or more corresponding residues. As used herein,these terms refer to the quantifiable similarity between amino acidsequences. One having ordinary skill in the art can compare amino acidsequences and calculate whether or not they correspond to each other.The terms “homologous”, “homology”, and “sequence similarity” are oftenused interchangably by those having ordinary skill in the art to referto corresponding amino acid sequences.

One having ordinary skill in the art can determine that an amino acidsequence corresponds to another amino acid sequences. The level of skillof those having ordinary skill in the art provides that amino acidsequences can be compared and sequence “similarity”, “homology”, and“correspondence” can be determined routinely. The processes of comparingand determining sequences correspondence are well known and widelyreported. See, for example, Bruck, C. et al., 1986 Proc. Natl. Acad.Sci. USA, 83:6578-6582, which is incorporated herein by reference. Onehaving ordinary skill in the art can construct a peptide having an aminoacid sequence which corresponds to another amino acid sequence.Corresponding amino acid sequences can be determined and peptides can beconstructed using other amino acid sequences as models. The amino acidsequence of such a peptide can be identical to that sequence from whichit was modelled. Peptides can be constructed that comprise amino acidsequences modelled after two corresponding sequences. An amino acidsequence can be determined which corresponds to both model sequences.

When the anti-idiotypic antibody is specific for an anti-receptorantibody, the specific portion of the receptor involved inligand/receptor interaction or pathogen/receptor interaction can beidentified. Peptides can be constructed which bind to the ligand orpathogen at the specific portion normally involved in receptor binding,thereby preventing receptor binding.

Harlow, E. and D. Lane, ANTIBODIES: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor NY 1988, which is incorporatedherein by reference, provide a review of the molecular and geneticaspects of mammalian immunology generally, and antibodies in particular.This publication contains a review of antibody structure and functionincluding variable regions and the CDRs thereof.

Antibodies bind to antigens by virtue of their secondary structure.Antibodies contains amino acid sequences within the CDR of the variableregion that form loops or reverse turns. The CDRs represent determinantstermed idiotypes. CDRs are quite variable in sequence. However, theirshapes are limited. Therefore, the number of conformations of the CDRsare limited. The peculiar shape or conformation of the CDRs isdetermined by a few amino acid residues. Therefore, one can imagineantibodies of a certain specificity as having a conserved beta sheetframework and loop projections which are generally comparable. Theindividual amino acid residues on each loop might be different when onecompares one antibody to another. The consequence is that a particularloops which bind antigens may vary in sequence from one antibody toanother but will often resemble each other in three dimensionalstructure. Since amino acid sequences of antibodies in their CDRs arehypervariable, they themselves can resemble foreign antigens which havesimilar loops containing similar amino acids. Accordingly, the means bywhich antibodies bind other proteins can be applied to constructimmunogens or mimetics of immunogens or biologically active proteins.

It is well known to those having ordinary skill in the art that thelight and heavy chains of antibodies contain variable regions and,within these regions, three loop portions known as CDRs which arehypervariable. The CDRs are the portions of the antibodies where bindingto epitopes takes place. That is, CDRs of an antibody comprise aminoacid sequences which form a three dimensional structure that directlyinteracts with the three dimensional structure formed by specific aminoacid sequences of the antigen to which the antibodies bind. A specificportion of the CDR loop interacts with a specific portion of an antigenmolecule. Methods of determining the amino acid sequence of the variableregions of antibodies are well known to those having ordinary skill inthe art.

According to the invention, identification of the amino acid sequence ofthe portion of antibody that reacts to a specific portion of the targetprotein can be used to construct biologically active compounds. It hasbeen discovered that anti-idiotypic antibodies contain regions ofpeptide sequences which correspond to the peptide sequences of theepitope for which the anti-pathogen antibody binds. It has beendiscovered that anti-receptor antibodies contain regions of peptidesequences which correspond to the peptide sequences of pathogen antigensor natural biologically active proteins which bind to such receptors.

Amino acid sequences of anti-idiotypic antibodies and anti-receptorantibodies which correspond to and which mimic epitopes and biologicallyactive portions of biologically active proteins, respectively, it hasbeen discovered that such corresponding sequences occur in the variableregions of the antibodies. The corresponding regions usually occurwithin the CDRs, often within the CDR II, that is the designated secondCDR. In particular, the corresponding sequences usually occur within oneor two of the CDR of the light chain and/or heavy chain. In the case ofanti-idiotypic antibodies, it has been discovered that portions of thoseantibodies which specifically interact with antibodies against anantigen correspond to portions of the antigen. Similarly, in the case ofanti-receptor antibodies, it has been discovered that they correspondto, that is, they share sequence similarity with, portions of pathogenantigens associated with pathogen attachment to cellular receptors andwith portions of biologically active proteins that interact withreceptors.

This technology is particularly useful to identify amino acid sequencesfor the following purposes.

1. To construct immunogenic compounds which mimic pathogen antigenneutralizing epitopes and are thereby useful to elicit neutralizingantibodies against pathogens, such compounds are useful as vaccinecomponents.

2. To construct biologically active compounds which block the binding ofpathogens to receptors on host cells and thereby prevent pathogenattachment which is usually essential in pathogen infection. In order toprevent pathogen/receptor interaction, such compounds can either bind tothe pathogen antigen that binds to the receptor or to the receptor.

3. To construct biologically active compounds which mimic biologicallyactive proteins by binding to the receptor sites of those cells, suchbinding causing alterations or effects to cellular function or behavior.Examples of such biologically active proteins include cytokines,hormones and growth factors.

4. To construct biologically active compounds which bind to the receptorsites of those cells to prevent or alter the effect which a biologicallyactive proteins would otherwise have upon those cells.

5. To construct biologically active compounds which bind to biologicallyactive proteins, preventing the biologically active protein from bindingto the receptor site on a cell, thereby preventing the protein fromcausing the effect which a biologically active protein would otherwisehave upon those cell.

The invention can be practiced by modeling compounds based uponsimilarity between pathogen antigens or biologically active proteins andthe CDR loops on the loop portions of the alpha helix of ananti-idiotypic antibodies or by modeling peptides based upon similaritybetween pathogen antigens or biologically active proteins andanti-receptor antibodies. In each case, the same result is achieved.That is, the identification of portions of an antibody sequence, a CDRloop, which are identical or similar to relevant significant regions ofa biologically significant protein; i.e. epitopes of antigens orbiologically active portions of biologically active proteins.

In the case of construction of immunogenic compounds which mimicpathogen antigen neutralizing epitopes, anti-idiotypic antibodiesspecific for antibodies against the pathogen antigen neutralizingepitope contain sequences corresponding to the pathogen antigenneutralizing epitope. The pathogen antigen neutralizing epitope can beidentified by comparing the amino acid sequence of the pathogen antigento the amino acid sequence of the anti-idiotypic antibodies,particularly the variable regions, particularly the CDR regions. Byidentifying which portion of the pathogen antigen contains theneutralizing epitope, compounds such as peptides can be synthesizedwhich are either identical or similar to the epitope of the antigen orto the region of the antibody. Vaccines can be formulated which includesuch compounds. These compounds will elicit a neutralizing antibodyresponse and immunity or protection from pathogenic infection will beconferred upon the subject of the vaccination.

In the case of construction of biologically active peptides which blockthe binding of pathogens to receptors on host cells, the amino acidsequences of pathogen antigens involved in such binding can beidentified by raising antibodies against the receptor and comparing theamino acid sequence of the pathogen antigen to the amino acid sequenceof the anti-receptor antibodies, particularly the variable regions,particularly the CDR.

Alternatively, anti-idiotypic antibodies specific for antibodies thatbind to pathogen antigens and thereby prevent binding of the pathogen tothe receptor can contain amino acid sequences that correspond to theamino acid sequences of the pathogen antigen which binds to thereceptor. The amino acid sequence of the pathogen antigen that binds tothe receptor can be identified by comparing the amino acid sequence ofthe pathogen antigen to the amino acid sequence of the anti-idiotypicantibodies, particularly the variable regions, particularly the CDRregions.

In either of these cases, by identifying the portion of the pathogenantigen that binds to the receptor, compounds can be synthesized whichare either identical or similar to the antigen sequence or to the regionof the antibody. The peptides can be administered to a patient. Thesecompounds will block a pathogen from binding to the receptor and therebyprevent pathogen attachment which is usually essential in pathogeninfection.

Pathogen binding to cellular receptors has been associated withalterations or effects on cell function and activity. In order toconstruct biologically active peptides which mimic the binding ofpathogens to receptors on host cells, the amino acid sequences ofpathogen antigens involved in such activity can be identified by raisingantibodies against the receptor which mimic the activity and comparingthe amino acid sequence of the pathogen antigen to the amino acidsequence of the anti-receptor antibodies, particularly the variableregions, particularly the CDR regions. Compounds can be constructedwhich are based upon the portions of both molecules that correspond toeach other, that is, that share sequence similarity. Such compounds willeither block the mimic the effect that pathogen binding has on cells orprevent pathogen binding from occurring and thereby prevent the effectscaused by pathogen binding.

It is contemplated that pathogen/receptor binding can be prevented byconstructing biologically active compounds which mimic the receptor siteand bind to the pathogen antigen. Such compounds are essentially “caps”to the antigen's receptor binding site and prevent the antigen frominteracting with the receptor. In order to construct such biologicallyactive compounds, the amino acid sequences of receptor site involved inpathogen binding can be identified by raising anti-idiotypic antibodiesspecific for anti-receptor antibodies that block pathogen binding andcomparing the amino acid sequence of the receptor to the amino acidsequence of the anti-idiotypic antibodies, particularly the variableregions, particularly the CDR regions. Compounds can be constructedwhich are based upon the portions of both molecules that correspond toeach other, that is, that share sequence similarity. Such compounds willmimic the receptor site and bind to the pathogen antigen at the receptorbinding site, preventing the pathogen from binding to the receptor.

In another embodiment of the invention, biologically active compoundscan be constructed by identifying the biologically active portion of abiologically active protein. The biologically active portion of abiologically active protein can be identified by generating antibodiesspecific for the receptor with which the biologically active proteininteracts. Such antibodies must either block the binding of thebiologically active protein of the receptor or mimic the activity of thebiologically active protein. The amino acid sequence of the biologicallyactive protein is compared to the amino acid sequence of theanti-receptor antibodies, particularly the variable regions,particularly the CDR regions. Compounds can be constructed which arebased upon the corresponding portions of both molecules, that is, thatportions that share sequence similarity. Such compounds will eitherblock the receptor or mimic the activity of the biologically activeprotein.

In another embodiment of the invention, binding of a biologically activeprotein to a receptor can be prevented by constructing biologicallyactive compounds which mimic the receptor site and bind to thebiologically active portion of the biologically active protein. Suchcompounds are essentially “caps” to the biologically active protein'sreceptor binding site and prevent the biologically active protein frominteracting with the receptor. In order to construct such biologicallyactive compound, the amino acid sequences of receptor site involved inbiologically active protein/receptor binding can be identified byraising anti-idiotypic antibodies specific for anti-receptor antibodiesthat block biologically active proteins from binding to the receptor andcomparing the amino acid sequence of the receptor to the amino acidsequence of the anti-idiotypic antibodies, particularly the variableregions, particularly the CDR regions. Compounds can be constructedwhich are based upon the corresponding portions of both molecules, thatis, the portions that share sequence similarity. Such compounds willmimic the receptor site and bind to the biologically active protein atthe receptor binding site, preventing the biologically active proteinfrom binding to the receptor and thereby neutralizing its ability toaffect cells. The essence of the invention is the discovery that thespecific portion of anti-idiotypic antibody or an anti-receptor antibodythat recognizes a neutralizing antibody or a receptor, respectively,corresponds to the neutralizing epitope of an antigen or thebiologically active portion of a biologically active protein whichnormally binds to the receptor, respectively.

The techniques needed to practice the invention are well known to thosehaving ordinary skill in the art. The starting materials needed topractice the invention are readily available.

Antibodies against a pathogen, a receptor or another antibody areproduced by routine methods. One having ordinary skill in the art candesign assays to determine whether an antibody is a neutralizingantibody. Such assays are well known and their design and operationroutine. Similarly, one having ordinary skill in the art can designassays to detect whether a pathogen is blocked from attaching to acellular receptor. Such assays are well known and their design andoperation routine. Furthermore, one having ordinary skill in the art candesign assays to determine the biological activity of a peptideincluding its ability to block the activity of another molecule are wellknown. Such assays are well known and their design and operationroutine.

Amino acid sequence determination can be readily accomplished by thosehaving ordinary skill in the art using well known techniques. Generally,DNA sequencing of relevant genetic material can be performed and theamino acid sequence can be predicted from that information. Sequencingof genetic material, including the variable regions of antibodies,particularly the CDRs, can be performed by routine methods by thosehaving ordinary skill in the art.

One having ordinary skill in the art can readily determine whether ornot one amino acid sequence corresponds to another. The determination ofwhether sequences are corresponding may be based on a comparison ofamino acid or nucleic acid sequence, and/or protein structure, betweenthe protein of interest, that is, the pathogen antigen, cellularreceptor or biologically active protein, and a member of theimmunoglobulin superfamily, in particular anti-idiotypic antibodies oranti-receptor antibodies, particularly the CDRs of the variable-regionsof such antibodies.

By determining the number of identical and conservatively substitutedamino acid sequences shared between two molecules, one having ordinaryskill in the art can determine whether or not two sequences correspond.The two sequences correspond if they share approximately at least 80%identical and conservatively substituted sequences of which at leastabout 28% are identical sequences and between about 30-42% conservativesubstitutions. Generally, corresponding amino acid sequences share atleast six similar amino acid residues. Corresponding sequences are oftenlonger, comprising about 10 or more similar residues. One havingordinary skill in the art, using routine techniques can byquantification determine whether two sequences are correspond within themeaning used herein.

Assays to determine whether or not antibodies are useful in a method toidentify biologically active peptides can be readily designed andperformed by those having ordinary skill in the art. Determination ofwhether an anti-pathogen antibody is neutralizing can be done by thosehaving ordinary skill in the art. Determination of whether ananti-receptor antibody mimics or blocks a biologically active proteincan be done by those having ordinary skill in the art.

Antibodies are generated against a pathogen by routine methods and, ifthey are found to be neutralizing, that is, if they prevent infection,anti-idiotypic antibodies are generated against the anti-pathogenantibodies. If the anti-idiotypic antibodies are capable of elicitingneutralizing antibodies, the anti-idiotypic antibodies are sequenced.Sequencing of the antibody can be directed at the variable regions,particularly the CDRs, by well known methods. The portion of the aminoacid sequence of the antibody that corresponds to an amino acid sequenceof the antigen of the pathogen is identified by sequencing both theantibody and the pathogen. The portion of the antibody where thesimilarity usually occurs is the variable region, in particular the CDR.A peptide is constructed which contains the amino acid sequence of thepathogen that corresponds to a portion of the anti-idiotypic antibody orwhich contains the amino acid sequence of the corresponding portion ofthe anti-idiotypic antibody. The peptide's ability to elicit aneutralizing antibody is confirmed. The peptide is useful in a vaccineto protect against infection of the host by the pathogen.

Antibodies are generated against a receptor that a pathogen binds to inorder to attach to a cell. An assay can be performed to determinewhether or not the anti-receptor antibody prevents the pathogen frombinding to the receptor. The portion of the antibody corresponding tothe antigen involved in receptor binding is identified by sequencing theantibody. Sequencing of the antibody can be directed at the variableregions, particularly the CDRs, by well known methods. The peptide issynthesized and will block prevent pathogen attachment to the receptor.The peptide is formulated as a pharmaceutic which is administered, forexample, as a therapeutic to combat pathogen infection.

Pathogens and biologically active proteins such as cytokine, hormonesand growth factors, bind to cellular receptors and alter the activity orfunction of a cell. Biologically active peptides are constructedaccording to the invention which, by binding to the receptor, mimic theeffect that pathogens or biologically active proteins have on cells.Alternatively, biologically active peptides are constructed whichprevent the binding of pathogens or biologically active proteins to thereceptor and thereby prevent or alter the effect those agents wouldotherwise have upon the cells.

Antibodies are generated against a receptor and selected for theirability to mimic the effect that pathogens or biologically activeproteins have on cells. If the antibodies are active, the portion of theantibody that is corresponds to either a portion of the pathogen antigeninvolved in receptor binding or a portion of the biologically activeprotein is identified by sequencing the antibody and the pathogenantigen or biologically active protein, respectively. Sequencing of theantibody can be directed at the variable regions, particularly the CDRs,by well known methods. The peptide is synthesized and will mimic thepathogen or biologically active protein. The peptide is formulated as apharmaceutic which is administered, for example, as a therapeutic toelicit the activity of that the native proteins have on cells.

In order to identify biologically active peptides which preventbiologically active proteins from binding to cellular receptors,antibodies are generated against the receptors. Antibodies that competewith biologically active proteins in binding to the receptor but that donot mimic the effect that biologically active proteins have on cells areselected. If the antibodies are block binding but are not active, theportion of the antibody that corresponds to a portion of thebiologically active protein is identified by sequencing the antibody andbiologically active protein. Sequencing of the antibody can be directedat the variable regions, particularly the CDRs, by well known methods.The peptide is synthesized and will block the biologically activeprotein but will not mimic its activity. The peptide is formulated as apharmaceutic which is administered, for example, as a therapeutic tocounteract the activity of the biologically active protein.

Biologically active compounds, such as peptides, can be constructedwhich mimic the binding site of the receptor and thereby bind to thebinding portion of either a pathogen antigen or a biologically activeprotein. Such peptides bind to the pathogen antigen or biologicallyactive protein, effectively preventing those proteins from binding tothe receptor. In order to identify biologically active peptides whichmimic receptor binding sites and bind to either pathogen antigens orbiologically active proteins, antibodies are generated against thepathogen antigens or biologically active proteins receptors.Alternatively, anti-idiotypic antibodies raised against anti-receptorantibodies can also be used. The antibodies are tested to identify thosethat prevent pathogen antigens or biologically active proteins frombinding to cellular receptors. Antibodies that compete with receptors tobind with pathogen antigens or biologically active proteins areselected. If the antibodies are block binding, the portion of theantibody that corresponds to a portion of the receptor is identified bysequencing the antibody and receptor. Sequencing of the antibody can bedirected at the variable regions, particularly the CDRs, by well knownmethods. The peptide is synthesized and will bind to either the pathogenantigen or the biologically active protein, thus preventing thoseproteins from binding to the receptors. The peptide is formulated as apharmaceutic which is administered, for example, as a therapeutic tocounteract the activity of the biologically active protein.

Peptides can be synthesized by those having ordinary skill in the artusing well known techniques and readily available starting materials.According to the invention, references to synthesizing or constructingpeptides is herein construed to refer to the production of peptidessimilar in sequence or structure to the corresponding regions identifiedby the method of the invention. These peptides may be produced using anymethod known in the art, including, but not limited to, chemicalsynthesis as well as biological synthesis in an in vitro or in vivo in aeukaryotic or prokaryotic expression system. The peptides may consist ofonly corresponding regions or they may comprise the correspondingsequences and addition sequences.

Peptides of the invention may be biologically active as produced or mayrequire modification in order to assume a three-dimensional conformationwhich is biologically active. Generally, the peptides are active asproduced. However, some modifications may be necessary for activity andsome modifications may be desirable to improve or alter activity.

Modifications which may be performed, using standard techniques,according to the invention include but are not limited to cyclization,disulfide bond formation, glycosylation, phosphorylation, or theaddition or subtraction of amino acid residues including amino acidresidues which serve to produce a useful three dimensional conformationvia a chemical linkage which is not generally found in natural peptidesand/or mimetics including but not limited to, those described inFreidinger et al., 1980, Science 210:656; Hinds et al., 1988, J. Chem.Soc. Chem. Comm. 1447; Kemp et al., 1984, J. Org. Chem. 49:2286; Kemp etal., 1985, J. Org. Chem. 50:5834; Kemp et al., 1988, Tetrahedron Lett.29:5077; Jones et al., 1988, Tetrahedron Lett. 29:3853.

Additionally, modifications may be performed, using standard techniques,according to the invention to create dimers or oligomers of the loops ormulti-looped structures.

An increase or decrease in bioactivity associated with modification maybe ascertained using the appropriate assay system. For example, if theactivity of the peptide is associated with immunogenicity, the abilityof modified and unmodified peptides to elicit an immune response may becompared.

Further, if the desired geometry of a peptide is known, computermodelling may be used to identify modifications of the peptide whichwould result in the desired geometry. The success of these modificationsin increasing bioactivity could then be evaluated using in vitro or invivo assay systems.

EXAMPLES

Example 1

The following embodiments of the invention are described in connectionwith experiments which have been conducted using reovirus types 1 and 3interactions with cellular receptors using the anti-idiotypeanti-receptor approach.

MATERIALS AND METHODS

Mice

Adult Balb/c female mice, 6 to 8 weeks to age, were obtained fromJackson Laboratories, Bar Harbor, Me.

Pre-immune serum was obtained on all mice used and assayed byneutralization of reovirus infectivity (see below) to ascertain thatthere had been no prior exposure to reovirus. Mice immunized withpeptides were housed in the animal care facility and fed a house diet adlibitum (Purina, St. Louis, Mo.). Mice immunized with reovirus type3/Dearing were housed in a separate facility.

Viruses

Reovirus type 1 (Lang), and reovirus type 3 (Dearing) and thereassortants 3.HA-1 and 1.HA-3 have been previously described (Fields,B. N. and Greene, M. I., Nature 20:19-23, 1982). Clones 1.HA-3 and3.HA-1 are single segment reassortant clones that segregate the S1 gene,the gene encoding the viral attachment polypeptide (hemagglutinin)sigma 1. For mouse inoculation and virus neutralization, a stock ofreovirus that was passed twice in L-cells was purified by substitutingultrasonic disruption (Branson Ultrasonic 200) for cell homogenizationin a modification of published techniques (Joklik, W. K., Virology49:700-715, 1972). The number of particles per ml was determined byoptical density at 260 nm (Smith, R. E. et al., Virology 39:791-810,1969).

Monoclonal Antibodies

Type 3 reovirus neutralizing monoclonal antibody 9BG5 (mouse IgG2aK)(Burstin, S. J. et al., Virology 117:146-155, 1982) was purified fromhybridoma supernatant with the cells grown in Dulbecco's minimalessential media (DMEM) (MA Bioproducts, Walkersville, Md.) with addedpenicillin/streptomycin solution (The Cell Center, University ofPennsylvania, Philadelphia, Pa.), and 10% fetal bovine serum (FBS).Culture supernatants were precipitated with 50% (NH₄)₂SO_(4,)solubilized in distilled water and dialyzed against three changes ofphosphate buffered saline (PBS). Next, the antibody was purified on aSepharose-protein A column and eluted with 0.1 M citric acid pH 4.5. Theeluate was collected in 1 M tris buffer, pH 8.5 to neutralize excessacidity and dialyzed against three changes of PBS. The dialysate wasconcentrated on an Amicon protein concentrator with a molecular weightcut-off of 30 kilodaltons (kD). The purified antibody was more than 95%pure by sodium-dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Irrelevant monoclonal antibodies UPC-10 and All (both mouseIgG2aK) were similarly purified from clarified ascites (Gibco, GrandIsland Biological Co.).

Monoclonal antibodies 87.92.6 (mouse IgM, K) and HO 13.4 (mouse IgM, K,anti-Thy 1.2) and HO 22.1 (mouse IgM, K, anti-Thy 1.1) were purifiedfrom 50% ammonium sulfate cuts of culture supernatant or from ascitessupernatants from ascites generated in hybridoma bearing Balb/c mice.These preparations were dialyzed against three changes of PBS and runover a goat anti-mouse IgM Affigel-10 column. Antibodies were elutedwith 3.5 M MgCl₂, dialyzed against three changes of PBS and concentratedas noted above. Purity of all monoclonal antibodies used was greaterthan 95% by SDS-PAGE.

Cell Lines

Murine L-cells were grown in spinner bottles with Joklik's MEM (MABioproducts) with 5% FBS. R1.1 cells (murine thymoma, Thy 1.2+) weregrown in suspension in RPMI 1640 (MA Bioproducts, Walkersville, MD)supplemented with L-glutamine, 10 mM HEPES buffer (MA Bioproducts) andpenicillin/streptomycin with 10% FBS.

Immunization of Mice

For the study of DTH response, groups of mice were inoculated witheither synthetic peptide or live reovirus type 3 subcutaneously (s.c.)in two separate sites on the dorsal flanks of the animal (over each hindlimb); 50 μg of a synthetic peptide or 10⁷ viral particles/0.2 ml weregiven in separate injections of 0.1 ml vol. Six days later, animals werechallenged in the left footpad with 3×10⁷ viral particles suspended insaline containing 2% gelatin (30 μl). Footpad swelling was recorded 24hr later in a blind fashion (Greene, M. I. and Weiner, H. L., J.Immunol. 125:283-287, 1980). Four animals per group were studied, andthe magnitude of the response was determined by comparing the challengedleft footpad to the untreated right footpad.

For the study of humoral immune response, mice were inoculated witheither synthetic peptide or live reovirus type 3 as above with thefollowing modification. The peptide was conjugated with chicken serumalbumin (CSA) as described below and 100 μg of the peptide conjugate wasinoculated s.c. in two divided doses. For mice immunized with syntheticpeptides, the first immunization was with peptide mixed with an equalvolume of complete Freund's adjuvant; whereas with subsequentimmunization the peptide was suspended in saline containing gelatin.Mice were immunized weekly for five weeks, and serum was obtained priorto the first inoculation, and then at the second and sixth week. Formice immunized with reovirus type 3, 10⁷ plaque forming units (PFU) wasinoculated s.c. on the first and third week.

Radioimmunoassay Procedure

The wells of 96 well V-bottom polystyrene plates (Dynatech Laboratories,Alexandria, Va.) were coated with peptide by diluting the peptides to 25μg/ml in distilled water and evaporating 50 μl in each well byincubating the plates overnight at 37° C. Wells were coated withreovirus type 1 or type 3 by diluting stock solutions of virus to4.8×10¹¹ particles/ml in 0.1 M NaHCO₃ pH 9.5, dispensing 25 μl per welland incubation overnight at 4° C. (London, S. D., et al. 1987).Following overnight incubation, peptide or virus coated wells werewashed three times with PBS and blocked with 200 μl/well of 1% gelatinin PBS with 0.1% NaHCO₃ by incubation for 2 hours at 37° C. The wellswere decanted, washed three times in PBS, and mouse serum or purifiedmonoclonal antibody was added, 50 μl/well, diluted in PBS containing0.5% gelatin and 0.1% NaN₃. Following a 3 hour incubation at 37° C., thewells were decanted, washed three times in PBS, and radioiodinated goatanti-mouse Kappa diluted in PBS 0.1% NaHCO₃ with 1 mg/ml chicken gammaglobulin was added, 100 μl =48,000 counts per minute (CPM) per well. Theplates were incubated overnight at 4° C., decanted, washed ten times intap water and dried under a heat lamp. Wells were then cut out using ahot wire and counted in a gamma counter. The CPM determined on blankwells not coated with. antigen is subtracted from CPM values determinedon antigen coated wells in all cases.

Fluorescence Activated Cell Sorter (FACS) Analysis

R1.1 cells (99% viability to trypan blue dye exclusion) were centrifugedand washed twice in PBS 0.1% NaN₃ with 1% bovine serum albumin (FACSmedia). Cells were resuspended at 10⁷/ml either in FACS media alone orFACS media containing peptide-BSA conjugates at 200 μg/ml. The cellswere incubated on ice for 45 minutes prior to addition of monoclonalantibodies from 0.5 mg/ml stock solutions to 100 μl aliquots to thefinal concentrations noted. Following an additional 30 minuteincubation, 500 μl of FACS medium was added to each sample, the cellswere centrifuged, washed once in 500 μl FACS media, resuspended in 10 μlFACS containing a 1:200 dilution of fluoresceinated goat anti-mouse Fab(Southern Biotechnology Associates) and incubated for 30 minutes on ice.500 μl of FACS media was added, the cells were centrifuged and washed in500 μl FACS media, resuspended in 200 μl FACS media and analyzed at theUniversity of Pennsylvania fluorescence activated cell sorter.

Neutralization of Virus Infectivity

The titer of neutralizing antibodies in serum sample were determined inthe following manner:

(i) Micro-neutralization: L-cells (5×10⁴ per well) were incubated in 96well dishes overnight at 37° C. Reovirus type 1/Lang (1/L) and type 3/Dwere serially diluted and incubated for 1 hour with the L-cells at 37°C. An additional 75 μl of MEM supplemented with 5% fetal bovine serum,1% glutamine was placed in each well. At 3 days following incubation at370°, the media containing virus was removed and the cells were stainedwith Gentian Violet (Gentian Violet 3.4 g/l, ammonium oxalate 8 g/l).The titer of virus used for neutralization was 4 fold in excess of thatquantity of virus that was lytic for the L-cell monolayer.

Reovirus type 1/L or 3/D at the appropriate concentration was incubatedwith an equal volume of mouse serum for 1 hr at 25° C on 96 well plates.The virus-serum mixture was then transferred to L-cell monolayers asabove. The titer of antibody was determined as the amount whichpreserved 70% of the monolayer as determined by visual inspection.

(ii) Virus plaque reduction: 100 pfu of reovirus type 1/L incubated for1 hour with L-cells (7×10⁵ cells per well) in 12 well Costar plates. Thetiter of virus in each well was then determined as previously described(Rubin, D. H., J. Virol. 53:391-398, 1985).

Synthesis of Peptides

Peptides were synthesized using a model 430 A Applied Biosystems PeptideSynthesizer (Applied Biosystems, Inc., Foster City, Calif.).Deprotection and release of the peptide from the solid phase supportmatrix were accomplished by treating the protected peptide on the resinwith anhydrous HF containing 10% anisole or 10% thioanisole for 1 to 2hr at 0° C. The peptides were then extracted with either ethyl acetateor diethylether and then dissolved in 10% aqueous acetic acid andfiltered to remove the resin. After lyophilization, the composition andpurity of the peptides were determined with both amino acid analysis andreverse phase high performance liquid chromatography. This procedure wasused for the synthesis of all peptides, including V_(L) and the variantpeptides of V_(L).

Conjugation of Peptides to Chicken Serum Albumin (CSA)

Prior to conjugating the peptides to CSA, the CSA was first derivatizedwith a nucleophilic spacer consisting of adipic dihydrazide, asdescribed by Schneerson, et al., J. Exp. Med. 152:361, (1980). 30 mg ofthe adipic dihydrazide-derivatized-CSA (CSA-ADH) in 5 ml 0.1 M sodiumbicarbonate was reacted for 15 min at room temperature with 7 mgm-maleimidobenzoylsulfosuccinimide ester (Pierce). To this reactionmixture was then added 50 mg peptide and the couples reaction was allowsto proceed at 25° C. for 2 hr. Following dialysis against 0.1 M ammoniumbicarbonate and lyophilization, the CSA-ADH-peptide conjugates wereobtained as dry white powders.

RESULTS

Determination of Shared Peptide Sequence

Prior work has shown that a monoclonal antibody denoted 87.92.6 raisedagainst monoclonal neutralizing anti-reovirus antibody 9BG5 mimics theintact virus by binding to cell-surface receptors specific for type 3reovirus. See Noseworthy, J. H. et al., J. Immunol. 131:2533-2538, 1983;Kauffman, R. S., et al, 1983 supra; and Co, M. S. et al., Proc. Natl.Acad, Sci. USA 82:1494-1498, 1985. Monoclonal antibody 87.92.6 competeswith reovirus type 3 for binding to specific cellular receptors therebymimicking the viral cell attachment protein sigma 1 (the viralhemagglutinin) in its binding domain. This domain is also implicated inthe neutralizing antibody response (Burstin, S. J., et al, 1982 supra;Spriggs, D. R. et al., Virology 127:220-224 1983). This implies that87.92.6 mimics the epitope on the hemagglutinin that interacts with thecellular receptor for reovirus.

The nucleic acid sequences of the heavy and light chain variable regions(V_(H) and V_(L) respectively) of 87.92.6 have recently been determined(Bruck, C. et al., Proc. Natl. Acad. Sci. USA 83:6578-6582, 1986), andthe sequences have been compared to that of the reovirus type 3 sigma 1protein (Bassel-Duby, R. et al., Nature 315:421-423, 1985). Inaccordance with the methods of the invention, shared sequence portionsof the antigen and anti-idiotype have been identified. Moreparticularly, a 16 amino acid sequence in the reovirus type 3 sigma 1protein encompassing amino acids 317 and 332 has been identified ashaving corresponding amino acid sequences to a combined sequenceencompassing the second complementarity determining regions (CDR II's)of the 87.92.6 heavy and light chain variable regions (V_(H) and V_(L)respectively). Specifically, amino acids 43-51 of the V_(H) sharesequence similarity with amino acids 317-324 of sigma 1 and amino acids46-55 of the V_(L) correspond to amino acids 323-332 of sigma 1 (Bruck,C., et al, 1986, supra).

In accordance with the methods of the invention, peptides correspondingto amino acids 317-332 of the sigma 1 protein 43-50 of the V_(H)sequence and 39-55 of the V_(L) sequence have been synthesized. Asdemonstrated hereinafter, immunization of Balb/c mice with thesepeptides results in neutralizing anti-reovirus type 3 antibodies andspecific cell-mediated immunity to reovirus. This establishes that thecorresponding sequences between the sigma 1 cell attachment protein andthe anti-receptor antibody predicts the neutralizing epitope on thereovirus hemagglutinin, sigma 1. This approach allows the rapiddelineation of neutralizing epitopes on pathogens and the development ofpeptide vaccines that elicit a neutralizing response.

Binding of Neutralizing Monoclonal Antibody 9BG5 to Peptides

The monoclonal anti-receptor antibody 87.92.6 binds to both the reovirustype 3 receptor and the neutralizing antibody 9BG5 (Kauffman, R. S., etal, 1983, supra). Applicants predicted that the peptides derived fromthe areas of similarity between 87.92.6 and the type 3 reovirus sigma 1protein (Bruck, C., et al, 1986 supra) would have similar properties.The peptides synthesized to test this hypothesis are shown in Table I.

The peptides used in this study were synthesized by solid-phase methodsas noted above. The sequences are shown aligned with maximum similarity.The amino acids marked with a closed circle are identical and thosemarked with an open circle are of the same. class. It will be noted thatthe tested peptides contain anti-idiotypic antibody residues in additionto the shared peptide sequence.

The reo peptide corresponds to amino acids 317-332 in the type 3 viralhemagglutinin. Computer modeling predicts this area to be predominantlya beta-sheet configuration and to include a beta-turn. The V_(L) peptiderepresents amino acids 39-55 of the light chain variable. region of87.92.6, and includes the second complementarity determining region (CDRII). Modeling predicts this area also to be a predominant beta-sheet andto include a beta-turn. The V_(H) peptide comprises amino acids 43-56 ofthe heavy chain variable region of 87.92.6 including CDR II of the heavychain. The control peptide, unrelated to this system, is also shown.

Based on these similarities in primary and secondary structures, it waspredicted that the reo and V_(L) peptides should be recognized byanti-reovirus type 3 neutralizing monoclonal antibody 9BG5. FIG. 1 showsthe results of a radioimmunoassay determining the binding of purifiedmonoclonal antibody 9BG5 to the wells of microtiter plates coated withthe peptides. To control for non-specific binding to the polystyrenewells, counts per minute (CPM) determined on blank wells not coated withpeptide is subtracted from CPM values determined on peptide coatedwells. In addition, since these peptides may also cause non-specificadherence of immunoglobulin molecules, the specific binding of theclass-matched irrelevant monoclonal antibody UPC-10 to peptide coatedwells and subtracted this value from those determined for 9BG5 wasdetermined. No significant binding was seen to the control peptide usedin this study. Similarly, binding to the V_(H) peptide only achievedbackground levels indicating that this epitope is not recognized by9BG5. There was a small amount of binding to the V_(L) peptide, whichhas strong similarity in its carboxy terminal sequence to the reopeptide carboxy terminal. Although slight, this finding was reproducibleon subsequent assays. Strong reproducible binding to the reo peptide by9BG5 was evident. Since 9BG5 is a neutralizing antibody, this datumimplies that the reo peptide contains the neutralizing epitoperecognized by 9BG5. The binding to the V_(L) peptide indicates that thearea of sequence between these peptides (amino acids 323-332 of thesigma 1 protein) is involved in the neutralizing epitope.

Binding of V_(L) Peptide to the Reovirus Receptor

Prior work indicated that the neutralizing epitope recognized by 9BG5 isinvolved in binding to the type 3 reovirus receptor (Kauffman, R. S., etal. (1983) supra; Noseworthy, J. H., et al. (1983) supra; Spriggs, D.R., et al. (1983) supra). It was therefore speculated that the V_(L)peptide might also interact with the viral receptor. To test thishypothesis the V_(H) and V_(L) peptides were coupled to BSA byincubating peptides and BSA in 0.1% glutaraldehyde followed by dialysisagainst PBS. These preparations were used to determine if 87.92.6specifically blocked binding to the type 3 reovirus receptor on R1.1cells. As shown in FIG. 2a, pre-incubation of R1.1 cells with V_(L)-BSAblocked the binding of 87.92.6 indicating interaction of V_(L)-BSA withthe reovirus receptor. This blocking effect is specific aspre-incubation of R1.1 cells with V_(L)-BSA had no effect on the bindingof HO 13.4, and isotype matched control monoclonal antibody that bindsto the Thy 1.2 molecule on the R1.1 cell surface (FIG. 2b). Theseobservations were consistently reproducible on multiple experiments. Anadditional control is shown in FIG. 2B where it is demonstrated thatV_(H)-BSA has no inhibitory effect on 87.92.6 binding when used at thesame concentrations as V_(L)-BSA. These data indicate a directinteraction of the V_(L) peptide with the reovirus type 3 receptor andimply that residues 46-55 of the 87.92.6 V_(L) chain and 323-332 of thetype 3 sigma 1 protein directly interact with the reovirus type 3receptor.

Binding of Reovirus type 3 Inhibits Host Cell DNA Synthesis UponReceptor Perturbation

Reovirus type 3 inhibits host cell DNA synthesis upon receptorperturbation. This effect is not due to infection of cells asreplication defective reovirus type 3 particles retain this property. Lcells were cultured at 5 ×10⁴ cells per well of 96 well microtiterplates in 100 μl media for 24 hours. Reovirus type 3 particles (A) wereadded and incubated for an additional 24 hours prior to the addition oftritiated thymidine. Purified monoclonal antibodies 87.92.6 or HO 22.1(B) were added for 1 hour at 37° C., then the culture media removed andreplaced with 100 μl fresh media for 24 hours, prior to the addition oftritiated thymidine. The cells were incubated for an additional 4-6hours and counts per minute (CPM) incorporated were determined.

FIG. 3 shows this effect of reovirus type 3 upon murine fibroblasts.Murine fibroblasts (which posses specific receptors for reovirus type 3)(L cells), were incubated with reovirus type 3, or left untreated (3A).Twenty-four hours later the DNA synthetic level was measured. Reovirustype 3 markedly inhibited DNA syntheses by these cells. 87.92.6 has asimilar effect on these cells, as shown in FIG. 3B. In this experiment,L cells were grown adherent and exposed to antibody for one hour, atwhich point the antibody was removed, and the cells cultured for anadditional 24 hours prior to determination of the DNA synthesis while acontrol antibody (HO22.1) had no effect. 87.92.6 similarly inhibits DNAsynthesis by fibroblasts, neuronal cells and lymphocytes.

Binding of Dimeric Peptides to Reovirus Type 3 Receptors

It was reasoned that V_(L) peptide may exhibit biologic effects similarto those exhibited by reovirus type 3 and 87.92.6. 87.92.6 is effectiveonly as a native antibody while monomeric Fab fragments have no effect.V_(L) peptide was synthesized with an additional amino terminal cysteineresidue (V_(L)SH) to form a dimeric peptide. V_(L)SH peptide wasdimerized by stirring a 5 mg/ml solution in 0.1 M ammonium bicarbonateovernight at 23° C. exposed to air. The peptides were then lyophilized.Dimerization was confirmed by Ellman determination according to theprocedure of Ellman, G. L. Arch. Biochem. Biophys. 74:443 (1958), whichrevealed less than 5% free sulfhydryl groups. L Cells were suspended at10⁶ cells/ ml in DMED 10% FBS and 50 μl added to each well of 96 wellmicrotiter plates. Following 24 hours of culture, peptides were added tothe concentrations noted, and the cells cultured for an additional 24hours. Tritiated thymidine was added for an additional 4-6 hours, andcounts per minute (CPM) incorporated was determined. Per cent inhibitionwas determined by the formula:$\left\lbrack {1 - \frac{\left( {{CPM}\quad {without}\quad {additive}} \right) - \left( {{CPM}\quad {with}\quad {additive}} \right.}{{CPM}\quad {without}\quad {additive}}} \right\rbrack \times 100$

The peptides utilized were:

V_(L):Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln

V_(L)SH:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln

Control: Cys-Tyr-Thr-Tyr-Pro-Lys-Glu-Asp-Thr-Ala-Asn-Asn-Arg

As shown in FIG. 4, marked inhibition of DNA synthesis was observed whenL cells were treated with V_(L)SH. V_(L) peptide monomers (without theadded cysteine residue) had no effect on L cell proliferation. Severalcontrol peptides utilized also had no effect in these assays (FIG. 4).This indicates that aggregation of the reovirus type 3 receptor on Lcells is essential for the inhibition of DNA synthesis by thesepeptides.

Down-Modulation of Reovirus Type 3 Receptor By Peptide Dimers

Aggregation of the reovirus type 3 receptor on some cells by 87.92.6leads to disappearance of that receptor from the cell surface. It wasreasoned that V_(L)SH peptide might similarly down-modulate thisreceptor. For these experiments we utilized murine thymoma (R1.1) cells,which have well characterized reovirus type 3 receptors were utilized.The effect of peptides on the level of expression of both the reovirustype 3 receptor (recognized by 87.92.6) and Thy 1.2 molecules(recognized by HO 13.4), as determined by flow cytometry was studied.R1.1 cells were cultured with peptides at the concentration noted (A),left untreated (B), or treated with 500 μg/ml peptide (C,D) for 1 hourat 37° C. The cells were centrifuged and washed three times in 1% BSA inPBS with 0.1% sodium azide (FACS media). Monoclonal antibodies 87.92.6(100 μl of affinity purified antibody) was added for 30 minutes on ice.The cells were washed and 100 μl of a 1:100 dilution of fluoresceinatedgoat anti-mouse Ig (Southern Biotechnology Associates, Birmingham, Ala.)was added for 30 minutes. The cells were washed and fluorescenceintensity analyzed by flow cytometry. Mean channel fluorescence wascompared for cells incubated in the presence or absence of primaryantibody to give mean channel fluorescence (FIG. 2B). Cells were stainedwith HO 13.4 (left panels in FIGS. 5A-D) which binds Thy 1.2 molecules,or with 87.92.6 (right panels in FIGS. 5A-D) which binds the reovirustype 3 receptor. Cells were treated with V_(H) peptide (FIG. 5A, leftpanel, and FIG. 5C) or V_(L)SH peptide (FIG. 5A, right panel, and FIG.5D).

The V_(H) peptide sequence:

V_(H): Cys-Gln-Gly-Leu-Glu-Gln-Ile-Gly-Arg-Ile-Pro-Ala-Asn-Gly

The other peptides are those described above for FIG. 4. As shown inFIG. 4, V_(L)SH peptide specifically down-modulates the reovirus type 3receptor in a dose-dependent manner, but does not effect the expressionof Thy 1.2 molecules on these cells. This down-modulation is a directbiologic effect of V_(L)SH peptide and not due to other factors in theexperimental design. The control peptide used (V_(H) peptide) does noteffect the level of expression of the reovirus type 3 receptor, or ofThy 1.2 molecules, on these cells. V_(H) peptide was derived from the87.92.6 heavy chain CDR II and does not specifically interact with thereovirus type 3 receptor. It has been demonstrated previously that V_(L)peptide in this form does not compete with 87.92.6 for binding to thesecells, although other forms of V_(L) peptide are able to inhibit 87.92.6binding. In addition, in the studies described in FIGS. 5A-D, the cellswere washed thoroughly to remove free V_(L)SH peptides prior to flowcytometry. Collectively these data indicate that competition for bindingto the reovirus type 3 receptor is not responsible for the decreasedstaining with 87.92.6. The down-modulation of the reovirus type 3receptor accounts for this phenomenon.

Receptor down-modulation is dependent on aggregation of the receptor, asdemonstrated in FIG. 6. Data from three experiments comparing the effectof V_(L) peptide monomers and V_(L)SH peptide is shown. R1.1 cells weretreated as described above with peptides (100 μg/ml) or 87.92.6 (a 1:1dilution of ascites), and analyzed for expression of the reovirus type 3receptor (87.92.6) or Thy 1.2 molecules (HO 13.4). Per cent decrease inmean channel fluorescence is calculated as follows: The mean channelfluorescence of peptide or antibody treated cells is subtracted fromthat of untreated cells, this divided by the mean channel fluorescenceof untreated cells; the resultant value is subtracted for 1 andmultiplied by 100. For peptide treated cells, mean channel fluorescenceis determined on peptide treated cells in the presence or absence ofprimary antibody. For antibody treated cells, mean channel fluorescenceis determined by the mean channel number of antibody treated cells inthe presence of primary antibody minus the mean channel number ofuntreated cells in the absence of primary antibody. Cells treated withantibody and then analyzed without primary antibody staining had anincrease in mean channel number when compared with untreated cells. Themean + standard deviation from 3 experiments is shown for peptidetreated cells. The peptides used in these experiments included:

V_(L):Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln

V_(L)SH:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln

Control: Cys-Thr-Tyr-Arg-Pro-Lys-Glu-Asp-Thr-Ala-Asn-Asn-Arg

V_(L) peptide monomers had no effect on reovirus type 3 receptorexpressions. V_(L)SH peptide specifically down-modulated the expressionof the reovirus type 3 receptor without effecting the expression of Thy1.2 molecules. The effect of V_(L)SH peptide was similar to that of87.92.6 (FIG. 6). The results indicate the specificity of the effect ofV_(L)SH peptide on the reovirus type 3 receptor and confirm thatreceptor aggregation plays a role in the induction of these effects.

Role of Specific Residues Of V_(L) Peptide Involved In The InteractionOf V_(L) Peptide With The Reovirus Type 3 Receptor

Once the shared regions were defined, variant peptides withsubstitutions at several positions in the putative binding domain ofV_(L) peptide were synthesized to study the effect of these forms of thepeptide on cellular physiology. These studies indicate that hydroxylgroups from positions 11 (Tyr), 12 (Ser), 14 (Ser) and 15 (Thr) may beinvolved in directly interacting with the reovirus type 3 receptor. Thisis the region of greatest shared identity of amino acids between theV_(L) peptide and the reo peptide. See Table 1. The variant peptides hadamino acid substitutions at positions 11-16, the region of the V_(L)peptide believed to be the binding domain. To study the effect of theseforms of peptide on cellular physiology, lectin induced mitogenesis wasutilized to provide a system wherein both receptor perturbation (by thepeptides) and aggregation (by the lectin) can be induced.

Peptide Inhibition of Lymphocyte Proliferation

Reovirus type 3 and anti-reovirus type 3 receptor antibodies have bothbeen demonstrated to inhibit concanavalin A (con A) induced lymphocyteproliferation (Nepom, J. T. et al., Immunol. Res. 1:255 (1982), Sharpe,A. H. and B. N. Fileds, J. Virol. 38:389 (1983), Fontana, A. and H. L.Weiner, J. Immunol. 125:2660 (1980)). The effects of these peptides onlymphocyte proliferation both in the presence and in the absence of conA have been investigated as follows.

C3H female mouse spleenocytes were prepared as a single cell suspension,and cultured with peptides at the concentrations noted in absence (A) orin the presence (B) of concanavalin A (con A) at 2.5 μg/μl. 72 hourslater, tritiated thymidine was added, the cells were harvested 18 hourslater and CPM incorporated determined. Per cent inhibition wascalculated as for FIG. 4. The peptides utilized are those described forFIG. 4. In the absence of con A, V_(L)SH peptide markedly inhibitedspontaneous lymphocyte proliferation, while V_(L) peptide had nosignificant effect (see FIG. 7A). How ever, in the presence of con A,V_(L)SH peptide and V_(L) peptide had similar effects in inhibitinglymphocyte proliferation (see FIG. 7B).

As shown in FIGS. 8A and 8B, when variant peptides were utilized lackinghydroxyl groups from positions 12 and 15 (V_(L)A12 and V_(L)A15respectively), the inhibition of con A induced lymphocyte proliferationwas attenuated (FIG. 8A). Lymphocyte proliferation was determined asdescribed above for FIGS. 7A and 7B. The peptides utilized were:

V_(L):Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln,

V_(L)F11:Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser-Gly-Ser-Thr-Leu-Gln,

V_(L)A12:Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala-Gly-Ser-Thr-Leu-Gln,

V_(L)A13:Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Ala-Ser-Thr-Leu-Gln, and

V_(L)A15:Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Ala-Leu-Gln

This indicates that these amino acid residues are involved ininteractions critical to receptor perturbation, leading to inhibition ofproliferation. The hydroxyl groups of positions 11 (Tyr) and 14 (Ser)appeared to have less of an effect on this cellular activity (FIG. 8B).

A peptide with a (Gly-Ala substitution at position 13 in the putativebinding domain of V_(L) peptide (V_(L)A13). Also utilized in contrast tothe other substitutions described, V_(L)A13 had an increased effect onthe inhibition of Con A induced lymphocyte proliferation at some of theconcentrations sed (FIG. 8B). This V_(L)A13 peptide also has increasedbinding to monoclonal antibody 9BG5, which may mimic the reovirus type 3receptor on these cells. These studies indicate that modification of theV_(L) peptide can identify specific residues required for receptorperturbation, and lead to the development of variant peptides with bothincreased and decreased biologic activity.

Competitive Binding of 9BG5 to 87.92.6 In The Presence Of Peptides

Polystyrene wells were coated with purified 87.92.6 or control IgM, Kantibody H022.1 by incubation of purified antibody (purified on a goatanti-mouse IgM column), diluted in 0.1% NaHCO₃ pH 9.5 to 1 μg/ml with 50μl/well, overnight at 4° C. The wells were washed, blocked with 2% BSAin PBS with 0.1% NaN₃, washed again and a mixture of radioiodinated 9BG5and peptides (at the concentrations noted in FIG. 9) were added for onehour at 37° C. The wells were washed and counted. In all cases, specificCPM bound was determined by subtracting CPM bound to blank wells coatedwith BSA from CPM bound for 87.92.6 coated wells. As shown in FIG. 9,binding of ¹²⁵I-9BG5 to wells coated with irrelevant mouse IgM, Kantibody H022.1 was similar to binding to blank wells. Per centinhibition was determined by subtracting specific CPM bound in thepresence of inhibitor from specific CPM bound in the absence ofinhibitor, dividing this by CPM bound in the absence of inhibitor, andmultiplying the result by 100. The ±SEM of values from two experimentsis shown in FIG. 9. V_(L) Peptide Inhibits Binding Of Reovirus Type 3Particles to 9BG5.

The wells of microtiter plates were coated with neutralizinganti-reovirus type 3 monoclonal antibody 9BG5 or irrelevant classmatched monoclonal A11 by adsorption to staphylococcal protein A (SPA).SPA (sigma Chemical Co., St. Louis, Mo.) was diluted to 5 μg/ml in 0.1 MNaHCO₃ pH 9.6 and 50 μl/well dispensed into 96 well polystyrene plates.Following overnight incubation at 4° C., the. wells were decanted,washed three times in PBS, and blocked with 2% BSA in PBS with 0.1% NaN₃for one hour at 37° C. The wells were decanted, washed three times inPBS and monoclonal antibody 9BG5 diluted to 10 μg/ml in 1% BSA in PBSwith 0.1% NaN₃ was added (50 μl/well) for 1-3 hours at 37° C. Priorstudies indicated that these amounts of SPA and monoclonal murine IgG2aantibodies gave maximal adsorption of antibody on the wells. The wellswere decanted and washed three times in PBS. Competitors were added atthe concentrations noted (100 μl/well) diluted in 0.5% BSA in 5 mMphosphate buffer with 0.45% NaCl and preincubated for 45-60 minutes at23° C. Control experiments indicated that these peptides had no effecton monoclonal antibody binding to the wells. Following preincubationwith inhibitors, radioiodinated reovirus type 3 particles diluted in 1%BSA in PBS with 0.1% NaN₃ were added (5-10×10⁵ CPM per well), and theincubation continued for 45 minutes. Wells were decanted, washed 8-10times with PBS and the CPM bound determined. V_(L) peptide inhibitsbinding of reovirus type 3 particles to 9BG5. As shown in FIGS. 10A-C,6,700 CPM were bound to 9BG5 coated wells and 500 CPM were bound tocontrol (A11 ) coated wells in the absence of inhibitors. The mean ±standard deviation of binding inhibition (Determined as noted for FIG.9) of replicate wells is shown. Control peptide B was used in thisstudy. The competitor peptides in FIGS. 15A and 15B are those describedherein. Competitor peptide V_(L)A6 is identical to V_(L) except thatalanine is substituted for asparagine at position 6. The competitorpeptides inhibited binding of reovirus type 3 particles to 9BG5.

V_(L) Peptide Inhibits Binding of Reovirus Type 3 and variant K to LCells

L cells were suspended at 10⁶ ml in 1% BSA in PBS with 0.1% NaN₃, and 50μl (5×10⁴) cells) added to each well of a 96-well microtiter plate, andpreincubated with inhibitors at the concentrations noted for 45-60minutes at 23° C. Equivalent input CPM of radioiodinated reovirus type3, type or variant K particles were added in 50 μl (700,000 to 1,250,000CPM/well) and incubated for 45 minutes. The cells were washed threetimes in 1% BSA in PBS with 0.1% NaN₃ and specific CPM bound determined,as noted in FIG. 9. As shown in FIGS. 11A and 11B, V_(L) peptideinhibits binding of reovirus type 3 and variant K to L cells. The mean ±S.D. percent inhibition of binding from replicate wells is shown versusthe final concentration of competitor. As shown in FIGS. 11C and 11D,V_(L) variant peptides also inhibit binding of reovirus type 3 to murineL cells.

Immunization with Peptides Induces Reovirus-Binding Antibodies

Having established that the V_(L) and reo peptides contain the epitopeinvolved in the interaction between type 3 reovirus and its specificcellular receptor, it was decided to test if immunization with thesepeptides would induce antibodies capable of interacting with reovirustype 3 and blocking infection. Groups of Balb/c mice were immunized withthese synthetic peptides as noted in the experimental proceduressection. Groups of 4 mice received either control peptide in adjuvant,V_(L) peptide coupled to chicken serum albumin (V_(L)-CSA) in adjuvant,V_(H) and V_(L) peptide coupled to CSA (V_(H)+V_(L)-CSA) in adjuvant,reo peptide in adjuvant or reo peptide without adjuvant. As a positivecontrol, an additional group of mice was injected with reovirus type 3.As indicated below, pre-immune serum from these mice disclosed noreovirus neutralizing antibodies indicating no prior exposure to virus.

Radioimmunoassay indicated a strong response to the immunizing antigenin all cases (data not shown). Binding of immune serum (day 60) toreovirus type 1 and type 3 is shown in FIGS. 12A-C. Specific binding wasdetermined by subtracted CPM bound on a blank plate from CPM bound on avirus coated plate. As a further control, specific binding of normalmouse serum to virus coated plates was also subtracted. To simplifyinterpretation, specific binding is shown for four groups of animals:those immunized with 1) the control peptide, 2) V_(L)-CSA, 3)V_(H)+V_(L)-CSA (all with adjuvant), and 4) reo peptide withoutadjuvant. Mice immunized with reo peptide plus adjuvant made a responsesimilar to those immunized with V_(L)-CSA, V_(H)+V_(L)-CSA plusadjuvant. Mice immunized with type 3 reovirus made a strong response totype 3 virus (specific CPM at a 10⁻³ dilution of serum of 10,428±807)with significant cross-reactivity with type 1 virus (specific CPM at a10⁻³ dilution of 6,976±915). As shown in FIGS. 12A-C, serum from miceimmunized with control peptide bound poorly to type 1 or type 3 viruscoated plates at any of the serum dilutions used. In contrast,significant binding of immune serum to type 1 and type 3 virus coatedplates is demonstrated from mice immunized with V_(L)-CSA,V_(H)+V_(L)-CSA or reo peptide. As was expected, binding to type 3 viruswas significantly higher than binding to type 1 virus, although somecross-reactivity is seen. The binding of type 1 virus was likely to havebeen due to some areas of primary sequence similarity between thepeptides used here and the type 1 sigma la protein (Manemitsu, S. M. etal., Biochem. Biophys. Res. Commun., 140:501-510, 1986).

These results indicate that priming mice with peptides modeled from theputative neutralizing epitope of type 3 reovirus or the correspondingepitope from the anti-receptor monoclonal antibody induces reovirusbinding antibodies.

Neutralization of Viral Infectivity by Immune Serum from PeptideImmunized Mice

Serum from peptide immune animals was assayed at three time points toevaluate its effects on viral infectivity of L-cells. Two assays wereused to detect neutralization of infectivity. One was a directcytotoxicity assay measuring the effect of serum on viral lysis ofL-cells grown adherent to the wells of 96-well microtiter plates byvital staining, and the other was by measuring inhibition of plaqueformation by serum, with virus and L-cells in soft agar. Results fromthe direct cytotoxicity assay are shown in FIGS. 13A and 13D. Pre-immuneserum from all of the animals used was assayed and no significant effecton type 1 or type 3 viral lysis of L-cells was demonstrated. As apositive control, neutralization of L-cell lysis by reovirus wasdemonstrated by serum from mice immunized with reovirus type 3. Thisserum produced potent inhibition of lysis by both type 3 and type 1virus, although a preferential effect on type 3 viral lysis was noted,with neutralization titers of 1:512 for type 3 virus on days 20 and 60,and titers of 1:342 and 1:256 for type 1 virus on days 20 and 50respectively. Serum from control peptide immunized animals had no effecton L-cell lysis by reovirus type 1 or type 3. Serum from mice immunizedwith V_(L)-CSA, V_(H)+V_(L)-CSA or reo peptide with or without adjuvantspecifically neutralized L-cell lysis by reovirus type 3 but not type 1(FIG. 13a versus 13 b). As results were similar for serum from animalsimmunized with reo peptide in the presence or absence of adjuvant,results only from the latter group is shown. This effect was also seenwhen serum from these mice was assayed for inhibition of plaqueformation. In FIGS. 14A and 14B, the reciprocal serum titer producing50% of greater plaque inhibition is shown for type 1 and type 3 virusfrom the groups immunized with V_(L)-CSA, V_(H)+V_(L)-CSA or reo peptidewithout adjuvant. Again, specific inhibition of plaque formation by type3 but not type 1 virus is seen. Since peptide-immune serum specificallyinhibits type 3 but not type 1 viral infectivity, these peptides definethe neutralizing epitope present on reovirus type 3.

Elicitation of Delayed-Tyne Hypersensitivity (DTH) to Reovirus Type byImmunization with Peptides

Prior studies have demonstrated that the specificity of DTH responses toreovirus infection involved the sigma 1 polypeptide (Weiss, H. L. etal., J. Immunol. 125:278-282, 1980. It was therefore determined ifimmunization of mice with these peptides would elicit DTH responses tointact reovirus. As shown in FIGS. 15A and 15B, significant DTHresponses to reovirus type 3 were induced by immunization with V_(L)peptide. This response was type specific as these animals did notdemonstrate significant DTH responses to reovirus type 1. Use ofreassortant viruses maps the response to the sigma 1 protein. Inaddition, priming animals with type 3 virus results in significant DTHto the V_(L) peptide. A type specific proliferative response to reovirustype 3 in spleen cells from mice immunized with reo peptide was alsodemonstrated. These data indicate that V_(L) and reo peptide define animportant epitope involved in T cell-mediated immunity to reovirus type3.

DISCUSSION

It has thus been demonstrated that synthetic peptides defined by areasof corresponding sequences between the reovirus type 3 sigma 1polypeptide and a monoclonal anti-receptor antibody 87.92.6 define theepitope on the virus and on the antibody involved in interacting withneutralizing antibody 9BG5, elicit neutralizing antibodies and induceT-cell mediated immunity. In addition it has been shown that one ofthese peptides, V_(L), competes with binding of 87.92.6 to the reovirustype 3 receptor on R1.1 cells. Since 87.92.6 competes with reovirus type3 binding to R1.1 cells (Kauffman, R. S., et al. (1983) supra), it ishypothesized that this epitope in the virus is involved in directlyinteracting with the type 3 reovirus receptor. This is confirmed by theability of V_(L) peptide to inhibit binding of reovirus type 3 to cells.Binding of reovirus type 1 (which utilizes a distinct receptor) is notinhibited, indicating a specific interaction with the reovirus type 3receptor.

Since this epitope encompasses amino acids 317-332 of the sigma 1polypeptide, this finding would seem at odds with other reports whichhave implicated amino acid 419 of the hemagglutinin in viral resistanceto neutralizing antibodies (Bassel-Duby, R. et al., J. Virol. 60:64-67,1986), and in tissue tropism of the virus (Kaye, K. M. et al., J. Virol.59:90-97, 1986). In those studies, viruses were selected for by growthin the presence of neutralizing antibodies (Spriggs, D. R., and Fields,B. N., Nature (London), 297:68-70, 1982), and those resistant toneutralization by the antibodies had their amino acid sequencedetermined (Bassel-Duby, R., et al, 1986, supra).

Several possibilities might account for the disparity in these results.It is possible that the mutations involving the amino acids 419 inducean allosteric effect on the conformation of amino acids 317-332 whichallows interaction with the viral receptor in the presence of theneutralizing. antibodies. In this scenario, amino acids 317-332 would bedirectly involved in binding to the viral receptor and to neutralizingantibody. The mutation at amino acid 419 would induce an allostericalteration in the confirmation of this region that would allow bindingto the viral receptor in the presence of neutralizing antibody. Anotherpossibility is that both regions are involved in binding the viralreceptor. In this case both regions would be in close proximity in thetertiary structure of the sigma 1 polypeptide. This is possible as bothare predicted to be in the “globular head” region of the hemagglutininby computer modeling (Bassel-Duby, R., et al, 1985, supra). The mutationof 419 would strengthen the interaction of this area of thehemagglutinin with the receptor, thereby overcoming the blockage ofreceptor binding by the neutralizing antibodies binding to residues317-332. While other possibilities exist, clarification of these issuesawaits more detailed knowledge of the tertiary structure of the sigma 1protein.

These studies have direct implications for vaccine development. It wouldbe greatly desirable to be able to delineate the neutralizing epitopespresent on microorganisms to aid in development of synthetic vaccinesthat would effectively protect individuals from infection, without therisks involved in the use of whole organisms. This would be particularlyuseful in situations where there is marked antigenic heterogeneity inthe structure of a pathogen, but the binding site for specific cellularreceptors is conserved. A variety of strategies can be and have beenemployed to determine sites involved in receptor-pathogen interactionsincluding site-directed mutagenesis and immunization of animals withsequential peptides derived from the sequences of pathogen products(Elder, J. H., et al, 1987, supra). Site directed mutagenesis, whileyielding specific information about sequence variations that lead todifferences in biological effects, suffers from the disadvantage thatallosteric effects resulting from the sequence differences could accountfor the effects induced. In this situation, sequence variation in aregion of a gene product may alter the biologic properties of a distantsite and yield misleading information. Analysis of the effects ofantibodies elicited by immunization with sequential peptides derivedfrom pathogen products, while a definitive approach yielding specificinformation, is time-consuming and may require analysis of a largenumber of peptides before a neutralizing immune response is detected.

The above experiments thus demonstrate a method for producing asynthetic biologically active peptide comprising a sequencecorresponding to a peptide sequence found in corresponding regions ofboth an antigen and in an anti-idiotypic antibody for that antigen. Bydemonstrating corresponding sequences in the sigma 1 cell attachmentprotein of reovirus type 3 and monoclonal anti-receptor antibody 87.92.6the neutralizing epitope of reovirus type 3 was localized. These studiesconfirm that the epitope implicated is the one involved in viral bindingto the cellular reovirus type 3 receptor and in the elicitation ofneutralizing antibodies. Once the shared region has been defined, otherbiologically active peptides can be prepared by modifying this peptidesequence. These modifications are directed to the region believed to beinvolved in binding of the antigen to the receptor. Gly(13) and hydroxylgroups from positions 11(Tyr), 12(Ser), 14(Ser) and 15(Thr) are believedto be involved in directly interacting with the reovirus type 3receptor. Peptide dimers comprising the shared peptide sequence alsohave biological activity and can be shown to have greater affinity thanmonomers.

As the studies herein indicate, modification of the V_(L) peptide canlead to development of variant peptides with both increased anddecreased biological activity. Peptide V_(L)A12 has reduced binding toneutralizing monoclonal antibody, reduced binding to the reovirus type 3receptor and reduced biologic activity. Peptide V_(L)A15 has increasedbinding to neutralizing monoclonal antibody, decreased binding to thereovirus type 3 receptor and decreased biological activity. Variantpeptides such as V_(L)A12, if used as immunogens, might prevent aneffective immune response. However, this might be clinically useful insome instances. V_(L) peptide itself, if used as an immunogen, mightelicit an effective immune response, but direct effects of the V_(L)peptide on the retrovirus type 3 receptor might be deleterious to thehost. In this case, a variant peptide such as V_(L)A15, which binds toneutralizing antibodies, but has reduced biologic activity, might beideal as an immunogen as it would elicit neutralizing antibodies butwould not be expected to have significant direct effects on theretrovirus type 3 receptor and would not be expected to be deleteriousto the host. The present approach of defining a shared peptide region ofboth an antigen and an anti-idiotypic antibody (anti-receptor antibody)for that antigen and subsequently modifying this peptide to producepeptides having more or less biological activity is believed to begenerally applicable to other receptor-ligand interactions.

The present approach further demonstrates a method of immunizing a hostmammal against an infectious organism having a site which bindsspecifically to a receptor site on a host cell. This method allowed forthe relatively rapid determination of the neutralizing epitope onreovirus type 3 and is believed to be generally applicable to otherpathogens for which neutralizing immune responses can be demonstrated.

In the present instance, the reovirus type 3 is known to selectivelybind to a structure which is antigenically and structurally similar tothe mammalian beta-adrenergic receptor. If attachment of a pathogen tospecific cellular receptors is important in the pathogenesis ofinfection by that pathogen, the approach outlined here should result inthe ability to determine the oligopeptide epitope involved in thepathogen-receptor interaction. This should also be applicable to otherreceptor-ligand interactions in a more general sense, and in the case ofpolypeptide ligands, should allow the determination of the bindingepitopes involved. It is believed that this strategy will lead to thedevelopment of biologically active compounds that will interact withspecific receptors in predictable ways. Accordingly, a method isdisclosed which is useful for synthesizing biologically active compoundsusing pathogen gene products, such as the reovirus 3, which is known tobind to a physiologic receptor of mammalian cells. Where, as with themammalian reovirus type 3 receptor, the result of such selective bindingis to affect the growth or other metabolic function of the subject cell,the subject method may be used for altering the growth of the mammaliancell by administering the synthetic peptide containing the subjectshared peptide sequence or biologically active modification thereof forthat purpose.

The strategy of utilizing shared primary structure and molecular mimicryto define interacting oligopeptide epitopes thus should have a widerange of applications in the biological sciences to both define areas ofspecific interaction between molecules, and to aid in the development ofcompounds with predictable biologic activity.

Those of ordinary skill in this art recognize that various modificationscan be made in the compounds of the invention without departing from thescope hereof. For example, peptides of the same class (i.e.,conservative substitution as described by Chu et al, “ConformationalParameters For Amino Acids in Helical, Beta Sheet, and Random CoilRegions Calculated from Proteins”, Biochemistry, 13(2):211, 1974, whichis incorporated herein by reference) may be substituted in the sequenceshared between the antibody and antigen, provided the activity of theresulting peptide is not adversely affected. Similarly, it iscontemplated that molecular modeling techniques will permit compounds ofquite different primary and secondary structures to be substituted forthe peptides of this invention, provided equivalent tertiary structures,as determined using the methods of this invention are employed.Additionally, other antibodies, such as other anti-receptor antibodiesto the reovirus type 3 receptor or anti-idiotypic antibodies toneutralizing antibodies may also contact the receptor using CDR regions.Peptides derived from these regions having biologic activity similar tothat described herein for V_(L) peptide are also within the scope of theinvention.

Example 2

The present approach also provides an alternative route for thedevelopment and production of biologically active peptides. As shown inFIG. 16, antibodies 15 specific for a receptor 11 of the antigen (orligand) 5 also mimic the antigen 5 in the same way as an anti-idiotypeantibody 9 of the antigen mimics the antigen 5.

In pathway I, an antibody 7 contains an epitope designated generally 21complementary to the neutralizing epitope designated generally 19 ofantigen 5. This antibody 7 is then used to produce other antibodies, oranti-idiotype antibodies 9. These anti-idiotype antibodies 9 will have aregion designated generally 23 mimicking the neutralizing epitope 19 ofthe antigen 5. In pathway II, the receptor 11 on cell surface 13contains an epitope designated generally complementary to theneutralizing epitope 19 of the antigen 5; the antibody specific for thereceptor 15 will thus contain a region designated generally 27 mimickingthe neutralizing epitope 19 of the antigen 5. The anti-receptor antibody15 is the equivalent of anti-idiotype antibody 9, since both containregions (23 and 27) mimicking the neutralizing epitope 19 of the antigen5. Anti-receptor antibodies 15 can be used as an alternative, or inaddition to, anti-idiotype antibodies 9 in the methods described hereinto develop and produce biologically active peptides 17 with propertiesof the antigen or ligand.

Because antigens such as viruses generally contain multiple antigenicepitopes, it may be necessary to screen the antibodies produced inresponse to the inoculation with the ligand, receptor or anti-ligandantibody to select antibodies having specificity for the neutralizingepitope of the antigen. Screening can be done by competitive assays thatdetermine the antibody's ability to inhibit binding of the antigen tothe receptor of the cell, those antibodies having a greater ability toinhibit binding of the antigen containing or mimicking the neutralizingepitope. Other screening methods include those as described herein inwhich a biological function, such as inhibition of DNA synthesis, istriggered. Suitable screening methods include those described herein,and in Burstin, S. J., et al., Hemagglutinin Virology 117:146-155. Itwill be obvious to those skilled in the art that various changes toreagents may need to be made in the competitive assays when differentantigen and receptor pairs are used.

As demonstrated herein, neutralizing antibody 9BG5, having a specificityfor the antigen HA3 on the reo virus, was used to make anti-idiotypeantibodies having anti-receptor activity. These anti-idiotype antibodiesalso bind to the reovirus type 3 receptor. The antibodies were screenedto identify antibodies that competed or inhibited binding of theneutralizing antibody with the receptor which would indicate theycontained epitopes that mimic HA3, the antigen. The variable region ofone antibody having this activity was compared with the sequence of theantigen HA3 to determine corresponding regions that define theinteraction site of HA3 and the receptor.

Instead of using an anti-receptor antibody that was produced as ananti-idiotype antibody, the receptor itself is also suitable forproducing antibodies that have epitopes mimicking the antigen. Toproduce antibodies by this route, receptor bearing cells are used as animmunogen, as for example in Drebin, et al., Nature (1984) 321:545-547and Drebin, et al., Cell (1985) 41:695-706. Alternatively, purifiedreceptor can be used, as for example in Williams, et al., 1989 J.Neurochem 53:362-369 and Meyers et al., 1992 Receptor 2:1-16, both ofwhich are incorporated herein by reference. These two immunogens can beused to make antibodies, usually monoclonal antibodies, by conventionaltechniques. An animal such as a mouse is first injected with thereceptor, its spleen cells are removed and fused with myeloma cells toform hybridoma cells, the latter are cloned in a serum-containing mediumand the monoclonal antibodies are separated from the medium. Theantibodies are then screened by neutralization assay, as describedabove, to select those antibodies which specifically bind to thereceptor site at the neutralizing epitope. This can be coupled with ascreen that examines the biological effects of receptor binding, forexample the inhibition of DNA synthesis assay described herein. In theexample, both the reovirus and the antibody cause some effect.

The strategy of utilizing shared primary structure and molecular mimicryto define interacting oligopeptide epitopes thus should have a widerange of applications in the biological sciences to both define areas ofspecific interaction between molecules, and to aid in the development ofcompounds with predictable biologic activity.

Those of ordinary skill in this art recognize that various modificationscan be made in the peptides and compounds of the invention withoutdeparting from the scope hereof. For example, peptides of the same class(i.e., conservative substitution as described by Chu et al,Biochemistry, 13(2):211, 1974, which is incorporated herein byreference) may be substituted in the sequence shared between theantibody and antigen, provided the activity of the resulting peptide isnot adversely affected. Similarly, it is contemplated that molecularmodeling techniques will permit compounds of quite different primary andsecondary structures to be substituted for the peptides of thisinvention, provided equivalent tertiary structures, as determined usingthe methods of this invention are employed. Additionally, otherantibodies, such as other anti-receptor antibodies to the reovirus type3 receptor or anti-idiotypic antibodies to neutralizing antibodies mayalso contact the receptor using CDR regions. Peptides derived from theseregions having biologic activity similar to that described herein forV_(L) peptide are also within the scope of the invention.

Example 3: Design of Immunogenic Human Immunodeficiency Virus Peptide

HIV, the AIDS virus, enters its target cell in a series of steps. Thefirst event in this sequence is the attachment of the viral envelopegp120 protein to CD4 on the surface of the target cell. In oneembodiment of the invention, gp120 is the protein of interest, that is,the pathogen antigen. Computer graphics and comparative molecularmodelling may be used to study the potential conformational propertiesof the CD4 binding site on gp120.

In the comparative modelling approach, the structure of an unknownprotein is deduced from sequence similarities between portions ofcrystallographically known proteins and the protein fragment to bemodelled (Greer et al., 1989, Prog. Clin. Biol. Res. 289:385-397). Thisapproach has been used in the modelling of a wide range of proteinsincluding antibodies and T-cell receptors (De la Paz et al., 1986, EMBOJ. 5:415-425; Chothia et al., 1988, EMBO J. 7:3745-3755). It has beenhypothesized that retroviruses are evolving towards structurallymimicking epitopes of the immunoglobulin (Ig) superfamily (Oldstone, M.B. A., 1987, Cell 50:819-820) that interact with normal immunestructures like CD4.

Structural examination of antigen combining regions of T cell receptorsand antibodies indicates that the recognition sites of this particularsuperfamily class are organized reverse turns or loops. For MHCmolecules, molecular recognition areas are highly α helical Bjorkman etal., 1987, Nature 329:506-512). Structural analysis of an anti-receptorantibody that mimics the cell attachment site of reovirus hemagglutininaffirms the possibility of shared β-type conformation as one underlyingrecognition feature bestowing mimicking properties on antibodies(Williams et al., 1988, Proc. Natl. Acad. Sci. USA, 85:6488-6492). Inthis context, an anti-CD4 antibody that competes with gp120 for the sameCD4 binding site is considered an anti-receptor (anti-idiotypic)antibody that mimics gp120 (McDougal et al., 1986, Immunol.137:2937-2944).

In the deduction of the possible topography for the CD4 binding site,one may first-examine the Protein Sequence Database (Devereux et al.,1984, Nucl. Acids Res. 12:387-395) using overlapping sequences of theBH10 isolate of HIV from residue 343 through 511. Optimal sequencealignment of the putative cell attachment site of gp120 with members ofthe Ig superfamily imply a degree of similarity between the site andantibody complementarity determining regions (CDRs) (FIG. 18).

The degree of similarity between the 383-455 region and CDR loopsimplies only that contact regions between gp120 and CD4 may exhibit βturns or loops, and not that gp120 itself folds like an immunoglobulin.This is evident because the intervening residues between the perceived βloops in gp120 are very different from those in antibodies. Thedisulfide bridge connecting residues 418 and 445 may preserve theanti-parallel β strands with a reverse turn geometry. In antibodystructures, CDR1 and CDR3 of light chains pack against each other andare stabilized by a disulfide bond. If this type of structure exists ingp120, then the β loop comprising residues 419-429 would be packed withthe β loop comprising residues 446-454; this packing may be stabilizedby the disulfide bridge formed between amino acids 418 and 445. In thismodel, residues 430-438, representative of a β structure, would still beexposed for contacting CD4.

A molecular model for the region 413-455 (FIG. 19) may be constructedbased upon these structural concepts, utilizing a light-chain antibodystructure known as REI (Bernstein et al., 1977, Mol. Biol. 112:535-542)as a template for the first and third hypervariable region, in which thecysteines in gp120 at positions 418 and 445 form a disulfide bridge. Thecysteines are positionally conserved with respect to each other as inthe light chain. The model may be energy optimized using molecularmechanics and dynamics. The model depicted in FIG. 18 indicates that theresidues 421-438 define a central turn region of the domain that may besurface exposed for interaction with CD4. Neutralizing antibodies havebeen shown to be directed toward this site (Sun et al., 1989, J. Virol.63:3579-3585). The in vitro biological activity of the 421-438 peptidefragment in modulating CD4-depending cellular function and thisfragment's ability to induce an anti-HIV response is as described below.This peptide was shown to block virus binding and appears to exhibitcellular regulatory functions such as immunosuppression that parallelsgp120.

The putative CD4 binding domain of the human molecule (Lasky et al.,1987, Cell 50:975-985) was modelled after an immunoglobulin variableregion. A topography of the purported gp120 binding site for CD4 wasobtained based upon a comparative modelling approach utilizingcorresponding sequences with the immunoglobulin superfamily(Kieber-Emmons et al., 1989, Biochem. Biophys. Acta 989:281-300).Molecular modeling of the 415-456 region of gp120 suggested that adisulfide bond can be formed by the cysteine residues at positions 418and 445. The model indicates that the residues 421-438 define a centralturn of the domain that is surface-exposed for possible interaction withCD4. Monoclonal antibodies that map to the 421-437 region have recentlybeen shown to have anti-HIV neutralizing ability (Sun et al., 1989, J.Virol. 63: 3579-3585). The 421-438 linear peptide termed B138 (Tables IIand III) was synthesized, purified, and assayed for its immunoreactivitywith human sera and its ability to induce an anti-HIV response.

The reactivity of linear B138 with sera (1:4 dilution) from healthyHIV-seronegative and seropositive study subjects has been assessed. Lowlevels of reactivity to B138 were detected using sera from nineteenHIV-seropositive, asymptomatic individuals, with only 2 of 18 (11%)generating levels of anti-B138 antibodies that were significantlydifferent (p<0.003) from HIV-seronegative controls. Thirty-three percent(7 of 21) of ARC patients and 10% (2 of 20) of AIDS patients hadsignificant levels of antibodies that bound B 138. Only one HIV-infectedindividual, an ARC patient, had levels of anti-B138 antibodies that weresignificantly different from HIV-seronegative individuals at 1:32dilutions of serum (p<0. 003). These results demonstrate that HIV-1 doesnot stimulate the production of antibodies that bind B138 in mostinfected individuals. Furthermore, in the few individuals that haddetectable levels of anti-B138 antibodies, these antibodies occurred atrelatively low titers and did not correlate with neutralizing orprotective activities.

The immunogenicity of B138 was determined by subcutaneous immunizationof mice and rabbits. Mouse antisera to B138 was shown to preferentiallybind to HIV_(HTLVIIB)-infected H9 cells as assessed by flow cytometryanalysis. Rabbit anti-B138 sera could immunoprecipitate purifiedrecombinant glycosylated gp120 as assessed by radiolabelledimmunoprecipitations, albeit weakly.

However, the immunogenicity of B138 increased dramatically when the B138was made into the cyclic peptide, 1005-45 (Tables II and III) bycysteine bond formation. Molecular dynamic calculations of the linear421-438 segment and the cyclic 418-445 fragment suggested that theconformational populations available to the 421-438 region under thecyclic constraints were restricted and more similar to our predictedgeometry of the gp120 putative CD4 binding epitope. Radiolabelledimmunoprecipitations (RIPs) of rabbit anti-sera against the cyclicpeptide showed significant specificity towards recombinant glycosylatedgp120 (FIG. 20). The reactivity is similar to that of theimmunoreactivity of polyclonal human HIV positive sera reactive withgp120. Infectivity assays with rabbit anti-B138 and rabbit anti-1005/45sera showed that both peptides could elicit neutralizing antibodies.

FIG. 21 shows the results of experiments in which rabbits were immunizedwith various gp120-derived peptides (see Table II) conjugated to Keyholelimpet hemocyanin (KLH) by glutaraldehyde fixation. Antisera wasobtained and tested for binding to recombinant HIV-1 gp120peptide-coated radioimmunoassay plates (using 1 μg/ml gp120 at 50μl/well, in 0.1 M NaHCO₃ and incubating overnight at 4° C.). Theindicator antibody was ¹²⁵I-labelled anti-rabbit immunoglobulinantibody. FIG. 21 indicates that the highest levels of bound antibodywere associated with antisera produced by animals immunized with 1005-45cyclic peptide as compared to lower levels of bound antibody associatedwith antisera produced by animals immunized with linear B138. Higherbound levels of antibody could result from a generally greater immuneresponse and/or the production of antibodies that have a higher affinityfor gp120.

The invention is not limited in scope by the embodiments disclosed inthe examples which are intended as illustrations of a few aspects of theinvention and any embodiments which are functionally equivalent arewithin the scope of this invention. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art and are intended to fall within thescope of the appended claims.

Example 4 As an alternative example, CD4 may be the protein of interest.

Interestingly, the CD4 receptor is only one of a small number of viralreceptors that have been cloned (White et al., 1989, Cell 56:725-728).An examination of the DNA sequence and the exon/intron organization ofthe nucleotides which encode the CD4 glycoprotein strongly suggests thatthis gene has evolved from a primordial Ig gene (see Littman, D. R.,1987, Annu. Rev. Immunol. 5:561-584 for review). Similarity profiles ofthe CD4 amino-acid sequence also reflect this relatedness to members ofthe Ig superfamily (FIG. 22; for reviews of the superfamily see Hood etal., 1985, Cell 40:225-229 and Williams et al., 1988, Annu. Rev.Immunol. 6:381-405). The most striking similarity between the CD4protein and the Ig superfamily occurs between the first 84 amino acidsof the CD4 glycoprotein and the Ig light chain variable domain (FIG.22). Although other CD4 regions also show similarity, secondarystructure predictions generated from this domain of CD4 are alsoextraordinarily similar to those observed for the Ig light chainvariable domains. Membership of the CD4 protein in the superfamilyimplies that the structure of the Ig-like domains of this protein shouldexhibit the classical “Ig-fold” (Schiffer et al., 1973, Biochemistry12:4620-4631; Poljak et al., 1973, Proc. Natl. Acad. Sci. U.S.A.70:3305-3310; Amzel et al., 1979, Annu. Rev. Biochem. 48:961-997).Therefore the alignment of CD4 with members of the Ig superfamilyindicates tertiary structural similarities when residues, identified byX-ray diffraction studies of antibody hypervariable regions, are alignedon the premise that the basic building-block structure and interactionhave been conserved (FIG. 23).

The sequence relationship between CD4 and immunoglobulins allows forgeneral conceptions about the structure of CD4 to be formulated andcorrelated with CD4 epitope mapping studies. Analysis of Ig structure todelineate possible unique epitopes may therefore be used to examinegp120 binding to CD4. Structural analysis of epitope locations on thesurfaces of antibodies suggests that there are separate ornon-overlapping (epitope) recognition sites that involve both classicalCDR and framework regions (Kieber-Emmons et al., 1986, Immunol. Rev.90:29-48; FIG. 23). Such regions have been referred to as idiotypedetermining regions (IDR) (Kieber-Emmons et al., 1986, Immunol. Rev.90:29-48). Each of these epitope (putative recognition) sites may haveunique functional properties. By inference, the CD4/Ig superfamilysequence alignment implies that HIV, MHC class II, ancillary proteinssuch as CD3, and T cell receptors may bind to CD4 in noncompetitiveways.

Example 5 Design of a Cyclic Peptide which binds to the CellularReovirus Receptor and can block the interaction between Reovirus and itsTarget Cells

In this embodiment of the invention, the protein of interest is animmunoglobulin molecule, and the method of the invention comprisesidentifying a region of the molecule which is similar to the CDR ofanother immunoglobulin, synthesizing peptides which comprise portions ofthe identified CDR, and then modifying the peptide such that it hasbiological activity. This example presents a nonlimiting working exampleof an embodiment in which a cyclic peptide is designed to resemble a CDRof an anti-virus receptor antibody.

MATERIALS AND METHODS

Peptides

All peptides were synthesized by solid phase methods, deprotected andreleased from the resin utilizing anhydrous HF. Peptides werelyophilized and further purified by high performance liquidchromatography utilizing a TSK 3000 column and lyophilized. Purity wasassessed by high performance liquid chromatography utilizing a C-6column and a 0-70% acetonitrile gradient. All peptides were greater than90% pure. Peptides (containing internal cysteine residues) were cyclizedfor experiments by dissolving them at 2 mg/ml in distilled water, andstirring them overnight exposed to the air. The peptides had no freesulfhydrlys following this procedure by Ellman determination.

Reovirus

Purified reovirus type 3 was prepared and radioiodinated using methodsset forth in Williams et al., 1988, Proc. Natl. Acad. Sci. USA85:6488-6492, which is incorporated herein by reference.

Monoclonal Antibodies

Monoclonal antibodies 9BG5, which binds to reovirus type 3hemagglutinin, and 87.92.6, which mimics the reovirus type 3hemagglutinin by binding to both 9BG5 as well as the reovirus type 3receptor, are as described in Williams et al., 1989, Proc. Natl. Acad.Sci. U.S.A. 86:5537-5541, which is incorporated herein by reference.

Determination of Free Sulfhydryls in Peptides (Ellman Determination)

Peptides dissolved in dH₂O at 2 mg/ml were added at 5, 10, or 20 μl to10 mM NaPO₄ pH 7.0 for a final volume of 1 ml. To this was added 6 μl of2,2′-bis azidothiobenzoic acid (ATBS, Sigma Chemical Co., St. Louis,Mo.) in 50 mM NaPO₄ pH 8.0. This was allowed to react for greater than 3minutes and the optical density (OD) at 420 nm was subsequentlydetermined.

Radioimmunoassay (RIA)

RIA plates (Dynatech Laboratories, Alexandria, Va.) were coated withpeptides by evaporation of varying amounts of peptides in distilledwater overnight at 37° C. The wells were washed with PBS, blocked with2% bovine serum albumin (BSA) in PBS with 0.1% NaN₃, and washed withPBS. Partially purified 9BG5 [(NH₄)₂SO₄ precipitate] was added atvarying dilutions for greater than 1 hour at 37° C. The wells werewashed in PBS and 50,000-100,000 counts per minute of ¹²⁵I-labelled goatanti-mouse light chain (anti-k & anti-[Sigma] iodinated by chloramine T)was added per well in 1% BSA in PBS for 1-2 hours at 37° C. The wellswere decanted, washed extensively, and CPM bound determined. SpecificCPM bound was determined by subtracting the CPM bound to uncoated wellsfrom the CPM bound to peptide coated wells.

Competitive RIA

RIA plates were coated with Staphylococcus protein A (Sigma ChemicalCo., St. Louis, Mo.) by incubation of 50 μl per well of a 5 μg/mlsolution overnight at 4° C. The wells were washed with PBS, blocked with2% BSA/PBS/0.1%NaN₃, and purified 9BG5 or isotype matched controlmonoclonal (All) adsorbed to the wells by incubation of 50 μl of a 10μg/ml solution (purified antibody) in 1% BSA/PBS/NaN₃ for 1-2 hours at37° C. The wells were washed, and competitors were added at variousconcentrations in 100 μl of 0.5% BSA/0.45% NaCl/0/05% phosphate bufferpH 7.2 for 1 hour at 37° C. 125I-labelled reovirus type 3 (5-10×10⁵ CPMper well) or unlabelled antibody (87.92.6 or isotype matched monoclonalE4.49.2) at a 1:100 dilution of ascites in 1% BSA/PBS/0.1% NaN₃) wasadded for an additional 30-45 minutes at 23° C. For wells incubated with87.92.6, the wells were washed in PBS, and ¹²⁵I-labelled goat anti-mouseIg added for an additional 60 minutes at 37° C. The wells were washedextensively, and CPM bound determined. For reovirus binding, specificCPM bound was determined by subtracting CPM bound to All coated wellsfrom CPM bound to 9BG5 coated wells. For 87.92.6 binding, specificbinding was determined by subtracting CPM bound following incubationwith E4.49.2 ascites from CPM bound following 87.92.6 incubation. %inhibition binding was determined by the formulae: [(Specific CPM boundw/o Inhibitor) - (Specific CPM bound with inhibitor)×100] / Specific CPMbound without inhibitor. That is, the amount of specific CPM bound w/oInhibitor minus the amount of specific CPM bound with inhibitor, thetotal amount remaining being multiplied by 100, the product of which isdivided by specific CPM bound without inhibitor.

Inhibition of Viral Binding to Cells

The cells were centrifuged and washed twice in 1% BSA/PBS/0.1% NaN₃.5×10⁴ cells or 1.25×10⁶ R1.1 cells in 50 μl were distributed in 2%BSA/PBS/NaN₃ blocked RIA wells. For peptide studies, 50 μl of inhibitorwas added in dH₂O to the cells. Following a 30 minute incubation, Lcells and ¹²⁵I-labelled reovirus type 3 were combined for an additional30 minutes at 37° C. The cells were spun, washed three times in ice coldPBS, and specific CPM bound was determined as noted above. Percentinhibition of binding was calculated by the formulae above.

Flow Cytometry Analysis

The ability of peptides to inhibit antibody binding to cells wasdetermined by preincubation of the cells with varying amounts ofinhibitor (in 100 μl dH₂O) for between 30 minutes and 1 hour at 23° C.Cells (either L cells or R1.1 cells) were washed in 1% BSA/PBS/0.1%NaN₃, and resuspended at 10⁷/ml. 100 μl of cells were then added in 1%BSA/PBS/0.1% NaN₃, and the incubation continued for 20-30 minutes.Antibodies (5 or 10 μl were added for an additional 20 minutes at 23° C.Ice cold 1% BSA/PBS/0.1% NaN₃, was added, the cells centrifuged andwashed prior to counterstaining with a 1:100 dilution of FITC goatanti-mouse Ig (Fisher Scientific) in 1% BSA/PBS/0.1% NaN₃. The cellswere washed twice and fluorescence intensity determined. Inhibition ofbinding was calculated as noted above with mean channel number utilizedin place of CPM.

Coupling of Peptides to KLH and Immunization

Peptides were coupled by glutaraldehyde fixation or specific couplingthrough a heterobifunctional cross-linker (MBS, Pierce Chemical Co.)(Romano et al., 1989, J. Neurochem. 53:362-369). Immunization was asdescribed in Romano et al., supra.

RESULTS AND DISCUSSION

Peptide Cyclization

One measure of the optimal folding conformation of V_(L) peptide isreflected by the ability of cysteine-containing variates to cyclize. Ifthe cysteine residues are placed in various positions in one or theother side of a predicted reverse turn, the residues placed in the mostenergetically favorable locations for assuming a reverse turn structureshould also cyclize most rapidly. This can be established utilizingseveral cysteine containing peptides as outlined in Table III.

These peptides were subjected to oxidation by agitating a solution (2mg.ml in 0.1 M NaHCO₃) at 37° C. for varying periods of time exposed toair. The disappearance of free sulfhydryls was quantitated by Ellmandetermination as above, and % loss of sulfhydryls with time calculated.The results are shown in FIG. 23.

Peptides (Table III) were agitated at 37° C. for varying periods of timeand loss of sulfhydryls quantitated. As noted, V_(L)C₆C₁₆ and V_(L)C₉C₁₆had the most rapid loss of sulfhydryls in this assay, while V_(L)C₁₀C₁₆peptide forms intramolecular disulfide bridges more slowly than theother two peptides, and implies that the corresponding cyclicconformation of V_(L)C₁₀C₁₆ may be energetically more costly to assumethan that of V_(L)C₈C₁₆ or V_(L) _(C) ₉C₁₆.

The oxidation of these peptides did not necessarily imply that cyclicpeptide formation was taking place, as intermolecular disulfide bridgesalso might have been forming. This issue was also examined by examiningreduced (2-mercaptoethanol treated) and non-reduced variates of thesepeptides by size-exclusion chromatography utilizing a Sephadex G-10superfine column. These studies indicated that both V_(L)C₈C₁₆ andV_(L)C9C16 peptides remained chiefly as monomers following oxidation,while a sizeable proportion of V_(L)C₁₀C₁₆ migrated more rapidlyfollowing oxidation. This indicates that the V_(L)C₈C₁₆ is formingintermolecular disulfide bridges, with subsequent formation of highermolecular weight forms. In contrast, V_(L)C₈C₁₆ and V_(L)C₉C₁₆ did notform intermolecular disulfide bridges, indicating that these peptidesmore readily fold into an appropriate conformation for intramoleculardisulfide bond formation.

Binding of 9B.G5 to Peptides

To assess the optimal conformation for binding of the V_(L) peptideanalogs, they were utilized to coat radioimmunoassay (RIA) plates, and9B.G5 bound by standard RIA procedures. The results are shown in FIG.24.

As can be seen, binding to V_(L)C₉C₁₆ peptide was higher than binding tothe other cyclic V_(L) peptide analogs. This indicates that V_(L)C₉C₁₆peptide has enhanced binding to 9B.G5 on solid phase RIA in comparisonwith the other cyclic peptides. Inhibition of 9B.G5 - 87.92.6Interaction by Peptides

While the solid phase RIA indicates a higher affinity of 9B.GS toV_(L)C₉C16 peptide compared to the other cyclic peptides, it does notaddress the affinity of this interaction in solution. To investigate theoptimal solution conformation for 9B.G5 binding, the peptides wereutilized to inhibit 9B.G5 - 87.92.6 interaction in a liquid phase assay.As shown in FIG. 25, the results of this assay indicate the V_(L)C9C16peptide again demonstrates a higher affinity of interaction comparedwith the other cyclic peptide variants. When compared with a linearanalog of V_(L) peptide (FIG. 26), V_(L)C₉C₁₆ peptide displays anincreased affinity of binding (40 fold higher affinity). This indicatesthat the increased conformational stability of this cyclic peptideincreases its binding affinity for 9B.G4. It is also demonstrated that apeptide derived from the 81.92.6 heavy chain variable region CDR II(V_(H) peptide) is able to inhibit the 87.92.6 - 9B.G5 interaction.While this peptide inhibits the idiotype- anti-idiotype interaction, itdoes not significantly interact with the reovirus type 3 receptor(Reo3R). Inhibition of 9B.GS-Reovirus type 3 Interaction by Peptides

To confirm that V_(L)C₉C₁₆ peptide represents an optimal conformationfor 9B.G5 binding in solution phase it was utilized to inhibit bindingof ¹²⁵I-labelled reovirus type 3 to 9B.G5 in a similar assay. Theresults are shown in FIG. 27. As can be seen, V_(L)C₉C₁₆ peptide alsoexhibited higher affinity than the other cyclic peptides in this assay.When compared with linear V_(L) peptide and dimeric V_(L)SH peptide(FIG. 28), V_(L)C₉C₁₆ peptide demonstrates higher affinity than linearV_(L) peptide, and similar affinity on a molar basis as dimeric V_(L)SHpeptide.

Inhibition of REO3R-87.92.6 Interaction by Peptides

To assess the affinity of the cyclic peptides for the reovirus type 3receptor (Reo3R), they were utilized in a series of assays to inhibitbinding of 87.92.6 or control antibodies to specific receptors. As shownin FIG. 29, V_(L)C₉C₁₆ peptide inhibited binding of 87.92.6 to murine Lcells and R1.1 thymoma cells. In contrast, linear V_(H) peptide had noeffect on 87.92.6 binding, while V_(L) peptide is a less effectivecompetitor on L cells, and ineffectual on R1.1 cells. The inhibition byV_(L)C₉C₁₆ peptide was specific as binding of isotype matched monoclonalH013.4 to Thy2 molecules was not inhibited by V_(L)C₉C₁₆ peptide (FIG.30). Thus, V_(L)C₉C₁₆ peptide is a specific Reo3R ligand with enhancedaffinity compared with its linear analog.

Inhibition of REO3R-Reovirus type 3 Interaction by Peptides

To further evaluate the interaction of V_(L)C₉C₁₆ peptide with theReo3R, the peptide was utilized to compete with ¹²⁵I-labelled reovirustype 3 for binding to the Reo3R. As indicated in FIG. 31, V_(L)C₉C₁₆peptide demonstrated higher affinity for the Reo3R than V_(L)C₈C₁₆peptide or V_(L)C₁₀C₁₆ peptide. When compared with linear V_(L) peptide(FIG. 30), V_(L)C₉C₁₆ peptide demonstrated 40 fold higher affinity forthe Reo3R, and similar affinity to dimeric V_(L)SH peptide.

This confirms that cyclic analogs of V_(L) peptide demonstrate higheraffinity of binding to the Reo3R than the linear peptide analogs. Thisstrategy should be applicable to peptides derived from other antibodyvariable regions, and defines an overall strategy for determining theoptimal conformation for binding of these peptides.

TABLE I Synthetic Peptides Comprising Corresponding Sequences of 87.92.6and the Reovirus Type 3 Hemagglutinin V_(H) Gln Gly Leu Glu Trp Ile GlyArg Ile Asp Pro Ala Asn Gly Reo Gln Ser Met --- Trp Ile Gly Ile Val SerTyr Ser Gly Ser Gly Leu Asn V_(L) Lys Pro Gly Lys Thr Asn Lys Leu LeuIle Tyr Ser Gly Ser Thr Leu Gln Control Lys Ser Gly Asn Ala Ser Thr ProGln Gln Leu Gln Asn Leu Thr Leu Asp Ile Arg Gln Arg

TABLE II Peptide Number Sequence 466 FRPGGGDMRDNWSEL 1005-45CRIKQFINMWQEVGKAMYAPPISGQIRC B138 KQFINMWQEVGKAMYAPP

TABLE III Peptides Utilized in These Studies Designation Sequence V_(L):    Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu GlnV_(L)SH: Cys Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser ThrLeu Gln V_(L)C₈C₁₆:     Lys Pro Gly Lys Thr Asn Lys Cys Leu Ile Tyr SerGly Ser Thr Cys Gln V_(L)C₉C₁₆:     Lys Pro Gly Lys Thr Asn Lys Leu CysIle Tyr Ser Gly Ser Thr Cys Gln V_(L)C₁₀C₁₆:     Lys Pro Gly Lys Thr AsnLys Leu Leu Cys Tyr Ser Gly Ser Thr Cys Gln B138:     Lys Gln Phe IleAsn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro 1005/45:     CysArg Ile Lys Gln Phe Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr AlaPro Pro     Ile Ser Gly Gln Ile Arg Cys

What is claimed is:
 1. A method of constructing a biologically activepeptide comprising the steps of: (a) comparing amino acid sequences ofcomplementarity-determining regions of an anti-receptor antibody thatmimics or blocks a biologically active protein that binds said receptorwith the amino acid sequence of said biologically active protein; (b)identifying an amino acid sequence of at least 6 amino acids in acomplementarity-determining region of said anti-receptor antibody thatcorresponds to an amino acid sequence of said biologically activeprotein; and (c) synthesizing a peptide which comprises said amino acidsequence of said anti-receptor antibody that corresponds to the aminoacid sequence of said biologically active protein; wherein said peptidebinds to said receptor and mimics said biologically active protein orblocks said biologically active protein from binding to said receptor.2. A method of constructing a biologically active peptide comprising thesteps of: (a) comparing the amino acid sequences ofcomplementarity-determining regions of an anti-receptor antibody thatmimics or blocks an antigen which binds said receptor with the aminoacid sequence of said antigen; (b) identifying an amino acid sequence ofat least 6 amino acids in a complementarity-determining region of saidanti-receptor antibody that corresponds to an amino acid sequence ofsaid antigen; and (c) synthesizing a peptide which comprises said aminoacid sequence of said anti-receptor antibody that corresponds to theamino acid sequence of said antigen; wherein said peptide binds to saidreceptor and mimics said biologically active protein or blocks saidbiologically active protein from binding to said receptor.